See also analytics & Characterization under antibody purification

Introduction to Mass Spectrometry

Mass spectrometry (MS) has emerged as a core technique for protein identificaiton and characterisation because of its high sensitivity, accuracy and speed of analysis. This development was largely due to advances in ionisation techniques, particularly matrix-assisted laser desorption/ionisation (MALD) and eelctrospray ionisation (ESI), which provided the gentle desorption/ionisation capability needed for the analysis of large biomolecuels without inducing enstensive thermal decomposition of the analyties. However, a drawback of MS is that it yeilds only the primary structure of individual proteins and cannot be used to study their behavior in solution or their oligoeric state . Oliva, Applications of multi-angle laser light-scattering detection in the analysis of peptides and proteins” Current drug discovery technologies, 1(3), pp. 229-242 (2004).

The common procedure for identification by mass spectrometry include (1) 2D-PAGE or other factionation procedure, (2) digestion with trypsin which are then (3) fed into either a MALDI-MS or a MS/MS or ESI-MSand (4) database comparison using algorithms.

For proteomics, one wants good data on peptide masses (MALDI-TOF MS) or good data that describes peptide fragmentation (ESI tandem MS). This requires sensitivity (amount of proteins capable of being detected), resolution (how well one can distinguish ions of very similar m/z values; usually need to be able to distinguish ions differing in m/z values of at least one Dalton (i.e., the mass of a single H atoms) and mass accuracy (as close as possible to the real value).

A mass spectrometer is an instrument employed to produce ions from atoms or molecules (the source) which are then separated according to their charge-to-mass (m/z) ratios and detected. In peptide mass fingerprinting, MS is used to measure the masses of proteolytic peptide fragments. The protein is then identified by matching the measured peptide masses to corresponding petpide masses form protein databases.

The way this all works is that trypitc digestion of each protein yeild a specific number of peptides of specific mass. The peptides then are analyzed by MS to obtain m/z values, which can be converted to masses. For example, tyrptic digestion of human hemoglobin alpha chain yeild 14 tryptic peptides, of which one petide has the mass of 1528.7348 Da. Thus, the singly charged ion of this peptide has an m/z value of 1529.7348. If wer were able to measure only to the nearest whole m/z value (i.e., 1529), there would be 478 matches. But the more accurate the m/z measurement provided, the fewer choices we would get. Accurate protein identifications usually also require multiple peptide matches. Even the best possible mass match would be unable to tell you whether the peptide you analyzed was from human or mouse hemoglobin. However, any real tryptic digestion would yeild multiple peptides and given you multiple m/z values to search against a database. (such as SWISS-PROT), OWL and NCBInr)

Here is another example: Suppose your peptide is AVAGCAGAR. Each residue in this has an amide NH group at one end, a C=O group at the other, and an alpha carbon with one proton in the iddle. The side chains give each amino acid its special chemistry are attached to the alpha carbon. Each amino acid has an average residue mass. For example, G has an average residue mass of 57.05. We would need to take on an extra proton (1 amu) to the N terminal residue and an extra OH (17 amu) to the C terminal. The cumulative nubmer of masses of each residue is how the MS sees this peptide.

When peptide ions collide with neutral gas atoms in the collision cell of a triple quad or ion trap (below), the kinetic energy they absorb induces fragmentation. In the most commonly observed cleavage, the bond between the carbonyl O and the amide N is cleave to form a “y-ion” and a “b-ion”. A y-ion is a fragment in which the + charge is retained on the C terminus of the original peptide ion and a b-ion is a fragment in which the charge is retained on the N terminal portion of the original peptide ion. Doubly charged ions are most likely to have charges at the opposite ends of the molecule. When these peptide ions fragment, both a b-ion and y-ion are formed. When singly charge ions fragment, either a b-ion or a y-ion is formed. The other half of the peptide is lost as a neutral fragment. One gets twice as much information from fragmentation of doubly charged as opposed to singly charged ions.

Lets take an example of glycine (C2H5O2N). As an uncharged residue, it has a mass of 75, as an ion (GlyH+) 76, as a fragemtn 57, as a b1ion 58, as a y1ion 76.

Ion Separation

Triple Quadrupole: consists of 4 meal rods arranged in parallel. Direct current and radiofrequency voltages are applied to the rods. Depending on the voltage applied, ions of a specific m/z value will pass through the quadrupole, whereas ions of greater or less m/z values will fly outwards an fail to pass through the quadrupole.

Ion Trap: Whereas triple quads analyze and peptide ions as they pass through the analyzer, ion traps collect and store ions in order to perform analyses on them. Ions collected in the trap are maintained in orbits wby a combination of DC and radiofrequency voltages. A particular ion of interest is selected and the trap voltages are adjusted to eject ions of all other m/z values. The voltages of the trap then are quickly increased to increase the energies of the remaining ions, which results in energetic collisions of the peptide ions with the helium gas atoms in the trap and induces fragmentation of the ions which are then caught in the trap and scanned out according to their m/z values.

Ion Detection: Typically detection using a conversion dynode which converts the ions into an electrical signal.

Ionization methods

Matrix-Assisted Laser Desorption (MALDI):

In a MALDI, 1) the sample to be analyzed is mixed with a chemical matrix which typically contains a small organic molecule with a desirable chromophore that absorbs light at a specific wavelenght, 2) the admixture of sample and matrix is then spotted onto a small plate/slide, 3) the target is placed into the source equipped with a laser which fires a beam of light at the target. The matrix chemicals absorb photons from the beam and become electronically excited. This energy is then transferred to the peptides or proteins in the same which are then ejected from the target surface into the gas phase.

This ionization process produces both positive and negative ions, depending on the nature of the sample. For peptides and proteins, the positive ions are usually the species of interest. The positive ions are formed by accepting a proton as they are ejected form the matrix. Each peptide molecule tends to pick up a single proton. For a peptide of say mass 1032, the addition of a proton and its one positive charge makes the m/z value 1003 for the [M+H]+ ion.

Electrospray ionization (ESI): In an ESI the sample enters the source through a flow stream (often from the HPLC) and passes through a needle held at high voltage. As the flow stream exits the needle, it sprays out in a fine mist of droplets. In contrast to MALDI, in which the sample is a dried, crystalline admixture of peptide sample and matrix, the peptides or proteins to be analyzed by ESI are in aqueous solution. Peptides exist as ions in solution because they contain functional groups whose ionization is controlled by the pH of the solution. Thus, carboxylic acids are protonated (unionized) below pH 3.0 and ionized at pH values above about 5.0. In contrast, N-terminal amines and histidine nitrogens are weak bases that are ionized below pH 7.0. The nitrogen functional groups of lysine and arginine are usually ionized below pH 8.5. This means that at acidic pH values (i.e., pH 3.5 and below), protonation of the amines will confer overall net positive charge to peptides and proteins. At basic pH, deprotonation of the amines and carboxyl groups confers a more negative overall charge.

A unique characteristic of ESI is the production of multiply charge ions from proteins and peptides. Many peptides bear multiple proton accepting sites and can exist as singly charge or multiply charged ions in solution. This is particularly true of peptides derived by tryptic digestion, as they bear lysine or arginine residues at their C termini as well as N-terminal amino groups, both of which may be protonated in acidic solution. Multiple charging serves the added purpose of forming ions that are within the mass range of quadrupole and ion-trap mass analyzers (below) which have more limited mass range than the TOF analyzers. For example, the absolute mass of a singly protonated 20 kDa protein (m/z=20,001) is well outside the mass range of a quadrupole mass analyzer which typically extends to 32 kDa of sometimes 4kDa. However, the typical 20 kDa protein will accept anywhere form 10-30 protons in solution. Thus for a population of these protein molecules in solution, some will have 20 protons and a m/z of 20,020/20=1001, some will have 21 protons and a m/z of 20,0021/21=953, and so on.

Three types of tandem mass analyzers are commonly paired with ESI sources. These are the triple quadrupole, the ion trap and the TOF discussed below. Those analyzers all perform the same type of analysis. From a mixture of peptide ions generated by the ESI source, the analyzers select a single m/z species. This ion is then subjected to collision induced dissociation which induces fragmentations of the peptide into fragment ions and neutral fragments. The fragment ions are then analyzed on the basis of their m/z to produce a product ion spectrum. which permits the sequence of the peptide to be deduced. The location of peptide modification also can be established.

Time of Flight (TOF); The ions formed in a MALDI source are extraced and directed into this analyzer. TOF measures the time it takes for the ions to fly from one end of the analyzer to the other and strike the detector. The speed with which the ions move is proportional to their m/z values. The greater the m/z, the faster they fly.

Having ions simply move from start to finish in a linear mode did not produce a good resolution (ability to distinguish ions of slightly different m/z values). This problem was solved with a reflectron

Surface enhanced laser desorption/ionization (SELD):

SELDI is a method of gas phase ion spectrometry in which the surface of substrate which presents the analyte to the energy source plays an active role in the desorption and ionization process. (US 5,719,060).

Glycosylation Analysis:

Site sepcific glycosylation analysis is a challenging taskt aht involves the termination of both the glycosylation site and the glycan microehterogeneity. One popular approach is to monitor the lgobal changes in clysoylation by releaseing glycans form a prtoein mxiture. The released glycan analysis however pvorides no protein specific information. Additionally quantitation of glycosylation on the site specific level is of great interest, but it is currently limited to gross comparisons due to the lack of glycan/glycopeptide standards. For relative comparison, glycan ion abundances are usually normalized to the most abundant glycan or the total glycan ion abudnance for relative comparisions. (Hong, “A mehtod for comprehensive glycosite-mapping and direct quantitation of serum glycoproteins” J of proteome research, 2015)

LC-MS/MS Analysis of glycopeptides:

Site specific clysoylation of may of the abundant glycoproteins in serum has been explored using different appraoches. A typical approach is the liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis of clycopeptides yeiled by specific prtoeases. An improvement invovles enrichment of the specifically glycosylated epptides frollowed by glycopeptide or rlreased glycan and peptide analysis. However, often these studies yield partial glycan heterogeneity or cinomplete site specific information. (Hong, “A method for comprehensive glycosite-mapping and direct quantitation of serum glycoproteins” J of proteome research, 2015)

Hong, (“A method for comprehensive glycosite-mapping and direct quantitation of serum glycoproteins” J of proteome research, 2015) discloses constructing comprehensive glycan maps of the common glycoproteins in serumplasma wehre each site is characterized for glycan hterogeneity. Both specific and nonspecific prtoeases, trypsin and Pronase E, were applied to obtain comprehensive site specific N-glycan maps of the top abudnant serum glycoproteins. An in house software too, GPFinder, was used to assign glycopeptides based on the LC-MS/MS analysis. The specificity of trypsin yeilds well defined peptides suitable for quantitation but may provide limited glycosie informaiton mainly due to alrge peptide sie or presence of multiple glycosites. Nonspecifi proteases usaully produce better glycosit coverage but yeiod variable peptide sequences that complicate the data analysi and limit its application to quantitation. Combining these tow approaches producesa more comprehensive glycap map with extensive site specifi c heterogeneity. The tyrpic map is then used to set the multiple reaciton monitoring conditions to monitor site specific glycoforms. 

Activatable Antibody Variant Analysis

HPLC-MS/MS:

Kavanaugh “A multi-analyte HPLC-MS/MS approach to assessing exposure of a probody Drug conjugate in precilinical studies” 2018) dicloses a protease activatable antibody prodrug (probody) targeted against CD71 (transferring receptor) and conjugate to a vcMMAE cytotoxic payload called “CX-2029”). In the intact, prodrug form, each light chain of CX-2029 contains an N-terminal prodomain which masks the target binding region of the parental antibody and decreases antigen binding. In vivo proteolytic cleavage of the prodomoin in the tumor microenvironment exposes the target binding region, yielding an active antibody. In vivo, CX-2029 may be present in serveral forms as a result of activation of the antibody prodrug as well as deconjugation of the cytotoxic payload. Kavanaugh developed a multi-analyte HPLC-MS/MS approach to monitor levels of four analytes: intact probody therapeutic, total probody therapeutic (present in both activate (binding) and intact (non-binding) molecules), probody-conjugated MMAE and unconjugated MMAE in mynomologus monkey plasma to evalute the exposure of both the intact CX-2029 and activate antibody prodrug. as well as to mnitor changes in the drug to probody ration over time. 

Western blotting: is used to identify a specific protein in a complex mixture of proteins. The technique exploits both the efficiency of SDS-PAGE to separate a mixture of proteins into distinct protein bands, adn the ability of immunochemical reagents to interact specifically with a given protein antigen. A typical Western-blotting protocol incluces the following steps:

(1) The mixture is first electrophoretically separated on a polyacrylamide slab gel in the presence of SDS.

(2) The protein bands are then transferred to a nitrocellulose membrane by electrophoresis.

(3) The membrane contianing the protein bands is serially incubated with (a) a suitable blocking reagent to prevent non-specific protein binding, (b) a wash solution to rinse any unbound blocking reagent (c) a probing antibody (anti-protein-of interest antibody) that forms a specific immune complex with Protein-of interst, (d) additional wash solution to remove any unbound antibody, (e) an enzyme-linked  that binds specifically to the Fc region of the anti-protein-of interest antibody, (f) additional wash solution to remoe any unbound enzyme-lined anntibody and finally, (g) a substrate solution, which int he presence of the enzyme, yields an insoluble, colored product that precipitates at the site of the immune complex, thereby rending the Protein of interest band visible.

Over the past decade, more than 30 organisms have had their genomes completely sequenced, including our own genome. But all of this information does not tell us what all the genes do, how cells work, how cells form organisms, how we age, what goes wrong in disease or how to develop a drug. This is where functional genomics comes into play. The purpose of genomics is to understand biology, not simply to identify the component parts.

DNA Microarrays

Among the most powerful tools for genomics are high-density arrays of oligonucleotides or parts of cDNAs. Nucleic acid arrays work by hybridization of labelled RNA or DNA in solution to DNA molecules attached at specific locations on a surface. Although it is possible to synthesize or deposit DNA fragments of unknown sequence, the most common implementation is to design arrays based on specific sequence information. 

DNA microarrays follows this general procedure:

• the chip itself with a special surface. Using technology from the semiconductor industry, manufacture begins with a 5 inch surface quartz wafer which is washed to insure uniform hydroxylation. 

• the device for producing microarrays by spotting the nucleic acids (probes) onto the chip. 

High-density Microarray (HDMs): In one highly used method, probe synthesis occurs in parallel resulting in the addition of A, C, T or G to multiple growing chains simultaneously. To define which oligonucleotide chains will receive a nucleotide in each step, photolithographic masks, carrying 18 to 20 square micron windows that correspond to the dimensions of individual features, are placed over the coated wafer. When ultraviolet light is shone over the mask in the first step of synthesis, the exposed linkers become deprotected and are available for nucleotide coupling. Critical to this step is the precise alignment of the mask with the wafer before each synthesis step. To ensure that this critical step is accurately completed, chrome marks on the wafer and on the mask are perfectly aligned. For more information click here

In these arrays, the so called oligonucleotide type microarrays, each gene is represented by multiple probe pairs with a bias toward the 3`end of the gene. Each probe pair consists of a perfect match and a mismatch oligonucleotide (that is, on average, 20 base pairs in length). The mismatch oligonucleotide contains a single base pair mismatch in the center of the probe. The mismatch is designed to measure background. The software subtracts the hybridization intensities of the mistmatch oligos from those of the perfect match oligos to determine the absolute or specific intensity value for each oligo set. Specifically, total or PolyA-selected RNA is used to create double stranded cDNA with a T7 RNA polymerase site incorporated. An in vitro transcription is run on this DNA, with biotinylated nucleotides included in the mix. This biotinylated aRNA is the target that is hybridized to the GeneChip®. During the washing and staining procedure streptavidin-phycoerythrin is used to make the biotin labeled aRNA flourescent. The chip is then scanned to capture an image of the fluorescence of each feature. The Affymetrix software measures the intensity of the signal from each perfect match probe, compares it to the signal for the mismatch probe, does this across all the pairs in a probe set for a given gene and, using a statistical algorithm, comes up with a call as to whether or not the gene is present in the original sample and a measure of the expression level if present. 

Spotted Microarrays (SMs)

In another methods, spotted arrays can be made from oligonucleotides as short as 30 mers, or clones as long as 2 kb. The DNA is put into a PCR plate and loaded onto the robot. The robot maneuvers pins to pick up the DNA solution and deposit it onto glass slides in precise predefined locations. To compare the gene expression between two tissue samples, cDNA from each sample is prepared using a separate florescent dye. The two samples are then mixed together and put on the microarray. Each spot on the microarray will indiscriminantly hybridize the corresponding cDNA from each sample. If one sample contained more mRNA of a certain gene than the other sample, then the microarray spot for that gene will emit a higher florescent signal when viewed under the appropriate light source. Using a high-resolution laser scanner and sophisticated software programs, the intensity in each channel for each spots is analyzed, revealing the difference in the expression level for that gene between the two samples. Numbers are relative and expressed as the ratio of expression level in the control sample vs an experimental sample.

Bead Arrays (BAs)

BAs do not involve planar substrates, but are comprised of an addressable population of microscopic polymer beads that contain precise amounts of up to four different fluorophores. Each type of bead has a characteristic DNA target suface coating where the identify of each tpe of bead is then determined optically by measuring the relative fluorescence from each fluorophore.

• labeled cDNA or cRNA targets derived from the mRNA of an experimental sample are hybridized to nucleic acid probes attached to the solid support. By monitoring the amount of label associated with each DNA location, it is possible to infer the abundance of each mRNA species represented. Although hybridization has been used for decades to detect and quantify nucleic acids, the combination of the miniaturization of the technology and the large and growing amounts of sequence information, have enormously expanded the scale at which gene expression can be studied

• a scanner is used to read the chips; 

• oftware programs to quantify and interpret the results. 

Applications and advantages of DNA microarrays include the following:

• Gene expression profiling (mRNA abundance): The transcription of genomic DNA to produce mRNA is the first step in the process of protein synthesis, and differences in gene expression are responsible for both morphological and phenotypic differences as well as indicative of cellular responses to environmental stimuli and pertubations. In terms of understanding the function of genes, knowing when, where and to what extent a gene is expressed is central to understanding the activity and biological roles of its encoded protein. 

• Entire genomes can be probed: It is no longer necessary to guess what the important genes are in advance. Now entire genomes can be included on a DNA microarray. Conventional methods such as northern and western blots or RT-PCR can still be used for measuring gene expression at the mRNA level as a follow up on genes that have been implicated by array results. 

Possible disadvantages to DNA microarrays are the following:

• Disparity between the relative expression levels of mRNA and their corresponding proteins.

• Posttranslational protein modification, protein-protein interactions and protein -DNA interactions are not taken into account.

Protein Microarrays

There are 2 general types of proteins microarrays: analytical and functional. Analytical microarrays involve a high-density array of affinity reagents (e.g. antibodies or antigens) that are used for detecting proteins in a complex mixture. Functional protein chips are constructed by immobilizing large numbers of purified proteins on a solid surface and have enormous potential in assaying for a wide range of biochemical activities (e.g., protein-protein, protein-lipid, protein-nucleic acid and enzyme-substrate interactions) as well as drug and drug target identification. 

Antibody Microarrays: are arrays of antibodies on a glass slide that bind specific antigens. A lysate is passed over the array and the bound antigen is detected after washing. Detection is usually carried out by using labeled lysates or using a second antibody that recognizes the antigen of interest.

• large range of protein expression: An example of this is where reagents might have high affinity for one protein but low affinity for another. Such reagents will still exhibit detection of the lower affinity protein if it is much more prevalent. Using sandwich assays can increase the specificity, however, but requires that at least 2 high quality antibodies exist for each antigen to be detected.

Possible disadvantages to consider with antibody microarrays are the following:

• requirement of detection antibodies which greatly limits the development of a higher density protein array system because it is very difficult to mix hundred or even thousands of antibodies together for high density protein arrays.
• possibility of cross-reactivity between antigens with an antibody.

Protein Chips: have become instrumental in learning about . These chips are constructed by immobilizing large numbers of purified proteins on a solid surface. Immobilization can be by absorption, covalent cross-linking and affinity

attachment. Applications and advantages of protein chips are the following:

• As one example, 5800 yeast ORFs were fused to GST-HisX6 at their NH2 termini and expressed in yeast using the inducible GAL1 promoter. The yeast expression strains contained individual plasmids in which the ORFs were fused in frame to GST. These fusion proteins were then printed onto glass slides using a commercially available microarrayer and then screened for their ability to interact with proteins and phospholipids. For example, the proteome was probed with a biotenylated protein of interest which was then detected suing streptavidin. (Zhu et al. Science 291:2101-2105,2001).
• Lam (US 5,510240) discloses a method of generating a random library of bio-oligomers such as peptides of a defined size and known composition attached to solid phase supports wherein each solid phase support is attached to a single bio-oligomer species and all possible combinations of monomer subunits of which the bio-oligomers are composed are included in the collection. The method includes the steps of completely coupling a subunit to substantially all sites of a solid support to form a solid phase support/new subunit combination. In the case of a peptide, for example, the coupling of the amino acids may be done by techniques well known in the art such as building a peptide from the carboxyl or C terminal end in which the C-terminal amino acid with its alpha-amino group protected is attached to a solid phase polymer. The protecting roup is then cleaved off and the next amino acid, also protected, is coupled by a peptide bond to the alpha-minao group of the amino acid attached to the solid support. The cycle of deprotection of the prior amino acid and coupling of the additional amino acid is repeated until the peptide is completed. In order to couple an amino acid to the growing synthetic chain, the carobxyl group of the blocked amino acid must be activated through methods known in the art. The biol-oligomer library can then be used to identify bio-oligoers within the library that demonstrate a biological activity of interest. 

Although there has been a lot of progress in the development of protein chips, proteins, by their nature, present many challenges for protein chips such as the following:

• lack of suitable amplification methods: Whereas one can use PCR to amplify DNA for DNA chips one must rely on different prokaryotic and eukaryotic expression systems to obtain proteins.
• Attachment to substrate: One challenge here is to make sure that the active site of the protein is not blocked due to the attachment.
• multiple forms of the same gene products due to posttranslational modifications, splice variants, etc.

Manufacturers of protein microarrays: Ciphergen Biacore Perkinelmer

Cell Microarrays

Cell microarrays use a microarray pin transfer device to transfer nanoliter volumes of a gelatin solution containing cDNA clones in expression vectors onto a slide. Transfection reagent and cells are then added, creating a cell microarray. Each array feature is a cell cluster overexpressing a specific cDNA. The array is then removed from media. The procedure is as following:

(1) nonoliter quantities of cDNA containing plasmids are dissolved in an aqueous gelatin solution and printed onto the surface of a glass slide using a robotic microarrayer device. 

(2) the printed arrays are then briefly exposed to a lipid transfection reagent, resulting in the formation of lipid-DNA complexes on the surface of the slide.

(3) mammalian cells in medium are added on top of the array in culture dishes, and cells that grow on top of the area where plasmids were printed take up these plasmids and become transfected. The result is a living microarray in which each feature is a cluster of 30-80 cells overexpressing a particular gene product. 

(4) to visualize cell microarrays, the array slides can be fixed and a variety of detection assays can be applied, including in situ hybridization, immunofluorescence and autoradiography.

Capillary electrophoresis with laser induced fluorescence (CE-LIF): is a method for the direct quantitation of gene expression. 

Detection and Quantification of Proteins Generally:

Gavin (US 2015/0093757) disclsoes capillary electrophoresis methods which include analysis and quantification of the amount of total protein in a sample. The method includes subjecting the sample to capillary electrophoresis in a separation matrix that includes a haloalkane compounds, exposing the separated sample to ultraviolet (UV) light for a sufficient period of time to react the haloalkane compound with tryptophan residues in the protein of the same to form fluroescent protein compoudns and detecting the fluroescence of the formed fluorescent compounds. In a further embodiment, the method includes detecting a specific target protein in a sample by performing capillary based immunoassay on the sample to detect the present or absence of the protein and detemining the results of the immunoassay to determine whether the protein is present or absent. The method includes icharged based (isoelectric focusing) and size (molecular weight based separation. The amount of protein is compared to other products in the sample to determine the relative amount of the protein. 

Roach (US 8,940,232 (ProteinSimple) discloses the use of capillaries having a small internal dimension such as a microchannelsfor condcuting electrophoretic or isoelectric focusing sepration, immobilization and fluorescence detection under automated control. Following sepration of the biologcial molecuels in the fluid paths of the capillaries with theri ionic charge by isoelectric focusing, the seprated molecuels are immobilized in their focused positions in the capillaries with ultraviolet light. 

Yang (US 9,400,277 (proteinSim;e) discloses methods for detecting analytes such as a protein in a fluid path which includes resolving asby isoelectric focusing (IEF), immobilizaiton as by photoimmobilzing one or more proteins in the capillary and detecting the protein(s) by contacting the photoimmobilized prtoeins with antibodies which can be labeled to form antibody-protein complex(es) in the capillary which are detected. In some embodiments, two or more detetion agents are used, one detection agetn for example a primary antibody that binds with one or mroe analytes and a second detection agetn, for example a secondary antibody, which can bind to the deteftion agent-analyte complex. 

Voss (US 2009/0023156) disclsoes methods for quantifying analytes which includes the steps of loading a microfluidic device, seperating the standard and the analyte by electrophoresis, immobilizing the standard and analyte, detecting the standard and anlyte with at elast one antiboy and comparing the signal of the standard to the signal of the analyte to determine the quantity of analyte in the sample. 

Detection of RNA:

Goldsmith (US7939252) dicloses a method of measuring an amount of ribo-nucleic acid (RNA) comprising incubating an RNA smaple with a fluorescenlty labeled RNA probe complementary to a sequence in a target RNA under conditions where the probe can hybridize to the target RNA to form an RNA-fluorescently labeled probe hybrid, passing the RNA-fluorescently labeled probe hybrid through a capillary electrophoresis system, detecting and recoridng changes in fluorescence as a peak as the RNA-fluorescenlty labeled probe passes through a detection window and determining an area under the peak, whereby the area under the peak indicates the amount of target RNA. 

Detection of Activatable Antibodies (Probodies): (see also Common Analytical Types under “Analystics and Characterization”)

Carman (US 2019/0117789) discloses activatable antibodies that bind CD166. The activatable antibody (AA) includes an antibody that binds to CD166 including a heavy chain and light change, a masking moeity (MM) coupled to the Ab wherein the MM inhibits the binding of the AB to the CD166 when the AA is in an uncleaved state and a cleavable moeity (CM) coupled to the Ab, wehrein the CM is a substrate that functions for a protease. In some embdoiments an agent such as maytansinoid is conjugated to the AA. The anti-CD166 conjugated activatable antibody was activated with matriptase or MMP14 for 2 hours at 37C and mixed with intact conjugated activatable antibody. The mixture was then analysed by the The WES Capillary electrophoresis system using anti-human IgG (American Qualex Catalog #A110UK). Carman showed the ability to separate matriptase-activated or MMP14 activated conjugated activatable antibodies form intact conjugated activatable antibodies. 

Desnoyers (US Patent Application No: 16/632265) discloses a method of quantitating a level of activation of an activatable antibody which includes the steps of 1) loading at least one capillary with a stacking matrix and a separation matrix. the “stacking matrix” refers to a highly porous (relative to the separation matrix) material that functions to concentrate proteins present in the sample and “stack” them at the interface with the separation matrix so that the proteins start migration under electrophoresis conditions form the same physical starting point. Suitable stacking matrices include those used to prepare stacking gels for Western blotting such as acrylamide, SDS. Capillaries pre-loaded with stacking matrix and separation matrix are available commercially as Wes Separation Module. Electrophoresis causes the compounds in the sample to migrate through the separation gel at differential rates according to molecular size/weight. 2) the HMW and LMW compounds are immobilzied within each capilalry as by using UV light. 3) Each capilarry is immunoprobed with a first reagent such as an idiotypic antibody that is specific for the activatable antibody. 3) Detection of the first reagent can be accomplised for example with a second reagent that binds to the first reagent. Exemplary detectably labeled second r agents include HRP-conjugate anti-mouse secondary antibody. 

Vasiljeva (US 2021/0025877; see also WO 2019/018828) disclsoes a method of quantitating a level of activation of an activatable antibody which includes constacting a loaded capillary with a biological sample which includes an antivatable antibody, an activated activatable antibody and combinations of both wherein the loaded capillary is preloaded with a stacking matrix (a highly porous material that fand a separation matrix , separating one or more HMW components from LMW components within each capillary, immobilizing HMW componetns and LMW components within each capillary, immunoprobing each cunctions to concetnrate proteins present in the sample and stack them at the itnerface with the separation matrix so that the proteins start migration under electrophoresis conditions form the same physical starting point. Suitable stacking matrices may be prpared form teh same materials used to prepare stacking gels for Western bloting such as acrylamide). apillary with at elast a first reagent that is specific for at least one activatable antibody and detecting and quantitating a level of the first reagent in each capillary. 

Mass spectrometry has become the method of choice for the Complex mixtures are separated before their MS analysis.

PCR: Principles of PCR BonesLab (degenerate PCR)    Boehringer (RT-PCR)    GeneHunter (Differential Display)    Competitive PCR Real Time Primer Sets RealTime Probe & Primer Database Ambion (Real Time PCR basics)

TRI REAGENT STEP

(1) centrifuge samples for 10 min at 10C at 1100 RPMI    (for adherent cells keep the 24 well plate and add tri reagent to the wells after you have taken off the supernatent. Use this trireagent for step 6)

(2) remove supernatant (throw down sink).

(3) Resuspend pellet with 1 ml PBS (using 1 ml pipette) and transfer into 1.5 ml tubes (the more rounded tubes in RNA cabinet) 

(4) centrifuge in small centrifuge (on work bench) (about 30 sec)

(5) remove PBS using vacuum (turn it on in room. Get asperiting pipette (from 2nd drawer and use regulat pipitte tips at end)

(6) Using Bulb pipette, add 1 ml Tri reagent (Sigma, from 3rd shelf) into 1.5 tubes using transfer bulb pipette (2nd drawer). (make sure pellet is resuspended)

(7) let sit for about 10 min

(8) place at -80C (freezer 2nd shelf)

RNA EXTRACTION STEP

(1) Set up Materials [RNA room: 1000, 200, 10 ul pipettes, all specially dept. chlorophorm and isopropynol) (figure how much will need and transfer into 15 ml tube (ex. 6 samples use about 2 ml) (Everything for RNA work should be kept together. This includes tips, racks, pipettes, etc). Clean the work area (something like “conflict” which is a little less aggressive than bleach, then “RNaseZAP” which should be left on surface for 10 sec) Wear gloves. Place 1 special PCR water (sigma) in warming plate and set at low)

(2)   Add 200 ul of chloroform to each sample using 1000 tips (otherwise you max out). (Transfer the amount of chloroform need from stock jar to a 10 ml tube rather than take from the stock jar) (ex. If 3 samples take abut 1 ml)

(3)   Vortex well until see strawberry like shake color.

(4)   Allow samples to sit for 2-15 min at rm temp until see clear layer at top

(5)   Prepare new set of 1.5 ml vials and label according to samples. Place samples in 2nd plastic holder and use this to transfer them to cold room

(6)   Centrifuge samples 15 min at 12000 rpms at 4C (cold room)

(7)   Add 500 ul of isopropanol into new 1.5 ml vials (again dispense from bottle to 10 ml tube 1st) (e.g, 2ml for 3 samples)

Precipitate the RNA

(8)   Transfer top clear layer (RNA layer) (using 200 ul pipette) (be careful not to get any pink layer) of sample tubes into isopropanol tubes. Vortex each tube after addition. (dispose of remaining pink layer in special waste container under hood)

(9)   Place at rm temp for 5-10 min (or -20C for 1 hr or at -70 overnight)

(10)                       Centrifuge 12000 for 15 min in cold room

(11)                       Look for pellet. Carefully remove most of supernatant (using vacuum with special glass tip and regular 200 ul tips. Add 1m of 75% ETOH (kept at -20). Do not disturb the pellet

(to prepare 75% ETOH. .75(100)=75 ml of 100 proof + 25 ml H2O (sigma)

(12)                       Centrifuge again 12,000 for 5 min at 4C (cold room)  (meanwhile prewarm water, see step 15)

(13)                       Remove all of the ETOH, using vacuum “RNA suckers” (non-yrogenic, RNAse/DNase free 200 ul tips, kept in RNA cabinet; orange box labeled “RNA suckers”; organge box labeled “RNA suckers”) (and when you get close to the pellet use pipetman with tips to move the ETOH away from the pellet)

(14)                       Allow pellet to dry with cap open for 2-10 min but not so long that pellet drys completely.

(15)                       Add 20 (40 for larger pellets or 10 ul for smaller ones) ul prewarmed sigma water (comes in a bottle which can be added to vials). Vortex to dissolve the pellet, Pulse spin down using spiner in RNA room and place in 55C warming block for 10 min (or you can freeze for further use) (use timer and after 10 minutes transfer samples to ice)

RNA Quantification

(16)                       obtain standard reagents (kept at +4C) and 2 new 15 ml tubes (from RNA cabinet) and 1 24 well plate + Sigma Water  Bottle (from RNA cabinet), ladder, black plate from RNA cabinet, RiboGreen RNA quantification kit (white top) 

(17)                       Prepare 1x TE Buffer  using TE calculator program (<my docs><Jim><TE calculator>)

Ex. stock solution will be 20x so need to dilute 20 times, so you can add 4.9 ml Sigma water and 0.1 ml 20 X TE in our case but this will vary on # of samples) (just insert # of sample in TE calculator on Disk which you can print out)

(To prepare new standards: (ex. 5 ml) We have 20x TE buffer in kit and want 1x.

1ul (TE):20ul (sigma water)   2ul:40 ul  3ul:60ul  4ul:80ul  5 ul: 100ul   so 50 ul for every 1000 ul sigma water  or more precisely 50 ul for 950 ul water   So 250 ul TE for 4750 ul sigma water

Obtain 4 new 2 ml (round btm tubes) and replace set according to directions on container. 1st add appropriate amounts TE with glass pipette. Then add appropriate amount from tube (ribosomal RNA standard marked with *) with pipette.

(18)                       Prepare Dye in 2nd tube by adding amount of dye (ribosomal quantification; blue cap) according to TE calcualtor program. 

Ex. add 1.6 ml 1x TE + 8 ul 200x dye stock)

(19)                     Take samples from warmer and put straight onto ice. Prepare 1:500 dilution of RNA samples in 24 well plate. Place 2 ul RNA in well and add 1ml TAE Buffer (1X) to give a 1:500 dilution [2:1000ul=1:500]. Shake the plate with hand. (you do not need to change glass pipette if not touching sides)

(20)                       Get black well plate (in RNA cabinet). Place black plate up at an angel. Add the following to the appropriate wells.

A1: (blank) 100 ul 1x TE only                 B1-E1: 100 ul standards

A2: 100 ul sample dilution 1, B2: 100 ul sample #2….                    Skip column 3

A4: 10 ul dilution sample 1 + 90 ul 1X TE: B4: 10 ul dil sample 2 + 90 ul 1x TE (This will give a dilution factor of 1:5000 because each of the samples are at a 1:500 dilution and by adding 90 ul TE to 10 ul of the sample, you are diluting by a factor of 10. So the 500 would become 5000.

1        2            3            4            5

Problem: We want 5 ml of 1XTAE buffer. How do we prepare from stock of 20x TAE. Answer: 1 +

19 ml H2O is 1X. But we want only 5 ml. So divide everything by 4. 1/4=0.25 ml + 4.75 H2O. 

(21)                       Add 100 ul of dye to each of the wells (including the blank) above. Doing this will change the concentrations of each well (the concentrations of the standards will be cut in half and the dilution factors increased 2 fold) as outlined below. (if you do not touch the sides can use single pipette tip)

1        2            3            4            5

(22)                       Sign up for fmax. Turn machin on (preferably 5 min before use). Open machin using icon on right top <M>. Place plate in fmax. Close dorr <D> icon to right and top. Open soft max program on desktop.  <file “cathy” <Ribog10.PDA> 

<template> Enter concentrations of standards under “sample” and “dilution factors” for samples if necessary.

<Read> 

Print out copy for records (select only page you need. ex. pg. 3-3)

exit program 

Reading will be in ng/ml so need to convert to mg/ml by moving the decimal 6 places to the left (eg., 10-9 ng becomes 10-6 ug).  The reading will enable you to determine how much of your RNA sample you will need to get 3 ug RNA. Dots next to #s mean dilution is wrong. 

Note: if there are (.) dots next to your numbers, you will need to choose other numbers because this means your #s are out of range (too concentrated). If all #s like this will need to dilute. Print page. Close fmax using icon top right.

(23)     Use the results to input into excell program that will tell you how much RNA from your sample to add to get 3 ug of RNA. (if 3 samples, input 4 on the excel program)

Sample                                RNA (ug/ul )                      RNA to Add [ul req. for 3ug]        Water to Add

DNAse Treatment (optional)

(1) label 0.5 PCR tubes according to # of samples

(2) Go to excell program (tells you how much RNA to add) Print out and hang up on hood

(3) add appropriate amounts to PCR tubes.

(4) spin down. Will get 2 layers 1 clear, 1 milky. Transfer clear layer (RNA w/out DNAse into PCR tubes (green top)

(5) leave in water bath for 45 min at 45C.

(4) Recalculate/quantitate amount of RNA as above

RT-PCR

(1)   clean hood with conflict 1st, then RNase. Obtain tips, bucket with ice, small ice rack (in RNA cabinet to hold tubes),Lable 0.2 ml PCR tubes and place in the tube ice tray.

(2) Use Excel spreadsheet from above to determine how much of the rest of reagents will need to add for master mix (in addition to the RNA and water above). You will need to add your # samples + one extra. Your total volume for the MM per sample is 10 ul composed of the reagents below.

(3) place your samples form the freezer in ice bucket.

(4) Get materials from PCR bucket (-20C) and dNTP from RT-PCR bucket.

(5) Lable an appropriate # 0.2 ml PCR tubes and place in the tube ice tray. Also get a small sigma water tube and 1.5 steril tube for the master mix.

(6) Add appropriate amount of H2O (calculation sheet next to “sample”) and sample RNA into PCR tube(s). Ex. for 2.90 ug/ul, add 1 ul RNA + 9 ul water) Pulse spin to get H2O to bottom.

(7) Place samples in thermocycler and perform 95 cycle (5 min) 

<turn on><files><load><95><enter><T=95.0 5:00 2. hold 4.0><close><exit><exit><start>

(8) Prepare Master Mix according to sheet (use total mix calculations).

EX.         For 6 samples you would get the following

Total Mix

(9) take PCR tubes with samples out of cycler and place on ice tray under hood.

(10) Add 10 ul of MM into each sample.

(11) Pulse spin tubes. Place tubes in thermocycler for 45 min (program called “CNRT” (45 min at 42C. Then 5 min at 95C (to stop rnct), then 4C hold). To start program press <exit><exit><start>(At this point you can stop and store your products at +4 up to several weeks or can continue)

PCR

(1) take out PCR excell sheet (prepare for # samples + 1 extra)

(2) Mix forward and reverse primers into 1 tube. (just tranfser 1 tube to the next and relable the tube as “Mix” and place primer name on top)

3) Label 0.2 ml PCR tubes and place in ice tray. Use different colored tubes for different primers. (bring bags with tubes out)., also get 2 tubes (1.5 for PCR) and another sigma water tube.

(4) Get the PCR materials (most of which are in PCR bucket) but dNTP is in the RT-PCR bucket.

(5) Prepare Master Mix below except that do not Add Ta1 yet. Again, you can prepare a program in Excel (can be the same sheet as above) to figure out how much of each reagent you will need for your mix. You can add everything except the primers to all of the tubes since everything else will be the same (just use same tip and dont touch) 

Ex. The total volume for each sample here is 23 and we have 6 samples. so:

for 6 samples (varies depending on rnct)

(6) Add 2 ul of your RT cDNA (from the thermocycler) to each new sample tube you prepared above. Also add 2 ul water instead of RNA to a separate tube.

(7) Now add your Taq to the to the master mix 

(5)   Add 23 ul of the master mix to sample tubes. Also add to neg control (the one with water instead of RNA)  pulse spin

(6)   Place tubes in thermocycler and run protocol (–turn on, place tubes in wells, lock in place, –file enter–load enter -CM60 enter) step 5: (6 to (28) varies depending on sample run. <exit><exit><start>)

PREPARE AGAROSE GEL

(1) obtain agarose from chemical shelf and TE buffer 1x. Get agarose beaker and top

(2) set up gel place by placing metal plates at ends.

(3) make 100 ml of 2% agarose. So 2 g agarose + 100 ml TE buffer (prepare TE buffer by adding 490 nonopure water _ 10 ml 50x TE)

(4) take the beaker to kellys lab and place in microwave on paper towel. heat 1 min (60 sec +start). Repeat 2 times and shake bottle in between heatings. Agarose should be fully dissolved

(5) cool beaker in ice bucket in sink filled with water (just hold beaker in bucket. don’t create air bubbles)

(6) Add 5 ul ethidium bromide using special pipette next to flask

(7) pour contents of beaker into gel plate (keep distance since carcinogenic!) If any air bubbles are created use pipette tip to move aside

(8) place 2 combs; 1 to left forward and one middle.

(9) wash flask immdiately with hot water first then distilled water. Turn upside down on paper in room

PCR THERMOCYCLER

Turn on cycler in back

Opening Files

(1) <Files><enter>

(2) <load><JRGRAD><enter> 

Saving Files

(1) <exit>

(2)<save><yes>

Setting the Gradient (to find the columns)

(1) <options><enter>

(2) <gradient><enter> (ex. T=50, G=5.0)(this means range 55-50-45) (each column will be diff temp; write down the column temps) (we use small blue tubes so small holes)

(3) Set your gradient temp and cycles

LID=(lid temp)

1) Initial denature T=92.0   3.00 

2) Denature T=94.0    15

3)Annealing          T=62 (hit the <opt> to condense step 3

4) Extension        T=72

5) Go To 2 Rep 39  (so this is really 40 cycles because already ran once)

6) Last extension    T=72

7) hold 4.0C   (when cycle completes move samples to refrig)

Load the Gel

(1) get 100 bp ladder from fezzer part. (will laod 5 ul ladder)

(2) Bring Big well plate forward (add 1x TE to plate if necessary to cover gel)

(3) Take well plate out of small plate, bring up in air and remove metal plates. Put the Gel plate under TAE buffer in large plate.

(4) remove combs. Get 96 well culture plate (use to laod samples) cut towel for place on bench and work on this.

(5) Load ladder (usually put ladder in center. load 5 ul) Usually leave end wells open.  If samples are not colored will have to use Dye (1:4; since 4X dye, use 4 ul dye for 16 ul sample). Mix the samples with dye in 96 well plate. If samples are colored can load directly 16 ul into wells. (use gel loading tips)

Load samples form left to right.  (ex. green tubes (p40) left and p35 right)

(6) run gel for 50 min at 100 volts or 45 min at 110 (select V for voltage and timer (45 min) then hit little runner)

Taking Picture of Gel

(1) connect leads to camera (power button on camera)

(2) turn camera on

(3) <kodack ID> icon (Kodack 1.D.3.5)

(4) <capture DC290>  (screen appears never change paremters. should say default. sample type: SYBRGREEN (gives better pciture) sample size: exposure time (3.5 sec)

(5) turn light on only for a few seconds while taking picutre

(6) <take preview> shut off light when picture comes on

(7) turn light on momentarily , Then <export image> (after a few seconds will hear click, then turn light off)

(8) save image

(9) <close>

(10) <rotate> picture b4 saving it

(11) <save as>

(12) can also take regular picture as back up.

(13) wash gel plate with distilled water.

Measuring Intensity with Kodak Machine

(1) <ROI “Region of interest”> icon on left        <2nd box (square)>        highlight 1st band (make box)

(2) <make a grid>    Ex. <colum-4> row=1  fro 3 bands. Hit arrow key so you can separate boxes away from each other

(3) read the intensity <show>    <ROI>    <analyze data>

(4) export data <file> <export data> <ROI>    <as displayed>    <put in folder>

(5) bring up saved program in excell <close program>    <excel>    <file>    <open>    <test files> since saved as text. text import wizard comes up    <next><next>    <finish>

(6) only interested in mean intensity so format the box <format><cell><#> 2 decimals. print out

Resuspending Primers

(1) Prepare 1X TE buffer. Make from 20X TE buffer RNase free small bottle at 4C. (Ex. you need 1 ml. so 950 ul sigma water and 50 ul TE buffer)  (Always transfer sigma water from the bottle to tube first)

(2) Add TE Buffer into vials containing primers. The Quality Assurance Document will tell you “nmol” This is the amount of TE to add.(ex. IL12p35F=52.3 ul).

(3) vortex and spin down

(4) from each vial take 10 ul and add 190 water. This gives a 1:20 dilution. [before use is 1 mM; we want 50 uM]  Dispesne these amounts into PCR tubes

(5) store at -80C (where the samples are kept) except that for 2 of the tubes (f and r) which you should combine (just transfer from one tube to the other) and relable this tube “mix” and date. This is working stock to be kept in freezer part of lab refrigerator.

Primer-DNA annealing temperature. This is generally dependent on the primer lenght and order and percentage of the nucleotides present composing the primers. 

Number of cycles (the number of times that the denaturing, annealing and synthesis process occurs).

Primer-dimers: The formation of primer dimers can inhbit the amplificaiton process because few or no primers are available to start the process. Once can avoid primer dimer formation by designing primers that do not complement each other, meaning that they bind to the biological agent’s nucleic acid and not to each other.

Primer Design: Primers and probes are generally designed for the detection of a given genus (genus-specific primers) or for the detection of a single species (species-specific primers). The 18S or 16S ribosomal RNA genes can be used for the design of genus-specific primers and probes because they contain sequences that are highly conserved between members of the same genus, but the sequences are variable among different genera. Species-specific primers and probes can be designed to target the ineranl transcribed spacer regions and intergenic spacer of the nuclear rRNA gene, because these genes are highly variable areas within a genus or among populations.

The similarity (homology) in the DNA of clsoely related species makes the design of specis-specific primers and probes difficult. GenBank can be used to find known DNA sequences. Using a sequence homology program like Basic Local Alignment Search Tool (BLAST) algorithm unique DNA regions can be found on the target sequence. A primer and probe design software package such as Primer Express (Applied Biosystems, Foster City, CA) can then be used in which parameters such as melting temperature, amplicon size, base content and primer/probe lenght can be selected. Validation studies are needed to ensure that there is no cross-reactivity with other species and to determine the lower detection limit of the assay with the primer and probe set. These studies comprise testing n umerous strains of the target organisms, as well as related and non-related microorganisms. In addition to specificty testing, the PCR should be optimized for maxzimum amplificaiton of DNA which involves testing various concentrations of the primers and other PCR reagents with DNA from the target organism.

Qauntitation is achieved through the amplifcation of standards containing known concentrations of DNA with real-tiem PCR. A range of 10 to 10e5 templates per reaction rpvoides a standard curve over five orders of magnitude. Automated QPCR systems provide sofware that will construct a standard curve of CT (the PCR cycle at which fluorescence is first detected) versus concentration. This # is inversely proportional to the concentration of the initial DNA template. The concentration of the unkown samples can be extrapolated form the standard curve by the sofware and reproted as the mean of two replicates.

Reagent storage: Avoid repeatedly freezing and thawing the reagents. This degrades nucleotides and primers.

Competitive PCR: is a a type of quantitative PCR. Exogenous target is added to create a competition between templates for amplification. The exogenous target is designed to amplify a smaller fragment with the same primer set so that products can be visualized easily by agarose gel electrophoresis and quantitated. The ratio of products obtained from exogenous and sample targets reflects the initial ratio of target to competitor.

DNA Fingerprinting Analysis: Amplification of regions of with sequence specific primers produces a banding pattern that can serve as a “fingerprint” to uniquely identify individuals. PCR-based restriction fragment length polymorphism (PCR-RFLP): The scheme of includes amplification of DNA containing the possible mutation or a known polymorphism using flanking primers, followed by restriction digestion of the PCR product. Banding patterns of the amplified fragments will show the mutation. Thus this PCR technique can be used to detect genetic mutations.

Long PCR: PCR reactions that are optimized for longer templates. This can be done by combining different DNA polymerases to increase processivity and fidelity of the reaction as well as increased extension times and the addition of cosolvents.

Multiplex PCR: PCR reactions where multiple sets of primers are used to amplify more than one target DNA. Thus, more than one unique target DNA sequence in a specimen can be amplified at the same time. Primers used in multiplex reactions must be carefully designed to have similar annealing temperatures, which often requires extensive empirical testing. Quantitative competitive PCR, a variation of multiplex PCR, can be used to quantify the amount of target DNA or RNA in a specimen. 

Nested PCR: Nested PCR, designed mainly to increase sensitivity (detect small quantities of taarget), uses two sets of amplification primers. One set of primers is used for the first round of amplificaiton, which consists of 15 to 30 cycles. The amplification products of the first reaction are then subjected to a second round of amplification with another set of primers that are specific for an internal sequence that was amplified by the first pirmer pair. The major disadvantage of the nested amplificaiton protocol is the high probabiltiy of contamination during transfer of the first round amplificaiton products to a second reaction tube. This can be avoided either by physically separating the two amplifcaiton mixtures with a lawyer of was or iol, or by designing the primer sets to utilize substantially different annelaing temperatures. 

Degenerate PCR: Instead of using specific PCR primers with a given sequence, you use mixed PCR primers with degenerate PCR. In other words, you insert “wobbles” in the PCR primer. The degeneracy of the primer is introduced during DNA synthesis.

Degenerate PCR has proven useful to find related genes in families.

RT-PCR: combines reverse transcription and PCR. This brings the benefits of PCR to analysis of RNA. In this process, RNA tragets are first converted to complementary DNA (cDNA) by RT, and then amplified by PCR.  RT-PCR can be used for amplification based cDNA library construction.

RNA Purification Kits: Gentra Systems

Differential Display: is a gene expression analysis method whereby mRNA from each sample is converted to cDNA which is then PCR-amplified using a combination of random primers (8-13mers) and anchored oligo-dT primers. The products are run on a gel. Each mRNA is represented as a signal band and differentially expressed bands are excised, cloned, and sequenced to reveal identity.

Ligase Chain Reaction (LCR): is a probe amplification technique. Successful ligation relies on the contigous positioning and correct base pairing of the 3′ and 5′ ends of oligonucleotide probes on a target DNA molecule. In the process, oligonucleotide probes are annealed to template molecules in a head to tail fashion, with the 3′ end of one probe abutting the 5′ end of the second. DNA ligase then joins the adjacent 3′ and 5′ ends to form a duplciate of one strand of the target. A econd primer set, complementary to the first, then uses this duplciated strand (as well as the original target) as a template for ligation. Repeating the process results in a logarithmic accumulation of ligation products, which can be detected by means of the functional groups atached to the oligonucleotides. 

Multiplex qPCR: refers to simultaneous quantification of multiple templates int he same reaction and depends on the ability to measure a different fluorescent signal for each nucleic acid target, often by using multiple TaqMan probes labeled with different fluorophores. One important applicaiton of multiplex RT-qPCR is the concurrent amplificaiton of a target gene and a housekeeping gene in the same reaction.

RACE PCR: amplifies 5′ and 3′ UTRs. For 3′ UTRs, one can use an oligo (dT) adapter linked primer to reverse transcript mRNA.

Real Time PCR: incorporates the ability to directly measure and quantify the PCR reaction products while the amplification is taking place. RT-rt-PCR has several advantages over conventional competitive RT-PCR. 1) it is up to 100 times more senstivie then conventional PCR; 2) it has a higher specifictiy because of three sequence specific oligonucleotides involved in the amplification process; 3) quantification is always performed in the log phase of PCR; 4) the linear range of quantification is wider; 5) there is almost no risk of cross-contamination with amplicons; 6) the detection of faulty amplifications is easy by software-based on-line verification of the overall quality of the PCR run. 

• intercalating dyes are typically used. SYBR Green is the dye of choice. It fluoresces 200 times more brightly when bound to dsDNA. Since SYBR Green will intercalate into any DNA in your reaction (ie., primer Dimers, contaminating DNA), you should do a melt curve analysis.

• Using Hybridization Probes using TaqMan and Molecular Beacons. The TaqMan 5NA (Perkin Elmer/Applied Biosystems) utilizes an oligonucleotide probe, whose sequence is selected by the investigator, double-labeled with fluorescent reporter and quencher dye molecules. As amplicons accumulate during the course of the reaction, the probe will hybridize to any target sequence present on the amplicons. When exposed to pulses of intense light, the fluorescent dyes located on the probe will respond with characteristic emission spectra which can be presented to the user as amplification plots. Because different probes can be labeled with different dye molecules, it is possible to simultaneously assay the same sample for the presence of different target sequences (i.e., “multiplexing”). In practical terms, this allows the same sample to be assayed for the presence of several different threat agents in a one tube reaction. 

Strand Displacement Amplification (SDA): is actually a non-PCR nucleic acid amplification technique developed in 1991. DNA polymerase initiates DNA syntheses at a single stranded nick and displaces the nicked strand during DNA synthesis. The displaced single stranded molecule then serves a a substrate for additional simultaneous nicking and displacement reactions. This isothermal DNA amplification procedure users specific primers, a DNA polymerase, and a restriction endonuclease to achieve exponential amplification of target. The key technology behind SDA is the generation of site specific nicks by the restriction endonuclease.

Touchdown PCR: involves decreasing the annealing temperature by 1 degree C every second cycle to a “touchdown” annealing temperature which is then used for 10 or so cycles.

Transcription Based Amplification System (TAS): includes synthesis of a DNA molecule complementary to the target nucleic acid (usually RNA) and in vitro transcription with newly synthesized cDNA as a template. Variations on this process are referred to as self-sustaining sequence replication (3SR), nucleic acid sequence-based amplification (NASBA) or transcription mediated amplification (TMA) Three enzymes, RT, RNase H, and T7 DNA dependent RNA polymerase are used in the reaction. Amplification steps involve the formation of cDNA from the target RNA by using primers containing a RNA polymerase binding site. The RNase H then degrades the initial strand of target RNA in the RNA-DNA hybrid after it has served as the template for the first primer. The second primer binds to the newly formed cDNA and is extended, resulting in the formation of double-strand cDNAs in which one or both strands are capable of serving as transcription templates for RNA polymerase. Although technically less robust and less sensitive than PCR, TMA has various merits that maike an attractive option: it works at isothermal conditions in a single tube to help minimize contamination risks.

Diagrams of PCR techniques

PCR is a way to amplify DNA sequences even when they are present at only very small quantities. It is a technique invented by Kary Mullis (U.S. Patent No. 4,683,202) PCR involves the use of oligonucletoide primers (flanking a gene segment of interest and orientated in a convergent manner such that polymerization proceeds in opposing directions on opposite strands), in conjunction with a heat-stable enzyme, e.g., Taq polymerase. By repeated cycling of the reaction through a range of temperatures permissible to the activity of the Taq polymerase, it is possible to synthesize large quantities of the desired gene segment to the exclusion of all other genes present in the sample. The newly synthesized gene segments, or DNA molecules, are referred to as amplicons and can be analyzed and viewed by electrophoresis on agarose gels. 

The steps to a PCR reaction are the following: (1) The DNA of interest is denatured into single strands using heat. (2) The DNA is then cooled in the presence of an excess of oligonucleotide primers which hybridize with the complementary ssDNA. Thus, this procedure requires that you know DNA sequences that flank the DNA sequence which you want to amplify. These flanking DNA sequences serve as your primers. (3) A temperature resistant DNA polymerase (Taq polymerase) is then added along with the 4 dntps. This will extend the primers forward and copy the DNA sequence from the 3′ end of the primer. The resulting DNA duplex is then separated by heating and the entire cycle is repeated. Since in each cycling there is a doubling of the DNA sequence, in just 25 cycles the desired DNA sequence can be amplified a million fold. The DNA can then be further characterized by southern blotting, restriction enzyme mapping and sequencing. 

Definitions

cDNA: is a single-stranded or double-stranded DNA molecule that complements a RNA molecule, preferably a mRNA. 

C(t) (threshhold cycle): is is the point at which the signal of a given reaction rises by some defined amount above the background signal level. 

Hot start: terms used to describe methods employed to avoid immediate start of transcription before denaturation and proper annealing of the primers by not allowing extension of the primers below a certain temperature.

Melting temperature (Tm): the temperature at which half of the hybrids are dissociated. As a general reule, the Tm is equivalent to the sume of 2C for each A-T base pair and 4C for each G-C base pair. Other factors that can affect the Tm are ionic concentration, pH, lenght of the complementary sequence, G-C content and mismatches (if any) between strands.

Taq DNA polymerase: T. aquaticus DNA polymerase (Taq polymerase) does not utilize RNA as a template so an initial RT step is required for PCR of RNA. Taq also has a 5′ nucleolytic activity, which hydrolyzes nucleic acid molecules hybridized to the template in its path. TaqMan is based upon the hybridization of a fluorescently labeled probe within the amplified sequence of a target DNA. A TaqMan probe is labeled with a fluorescent reporter molecule at the 5′ end and with a quencher molecule at, or near, the 3′ end. When the probe is unbound in solution, reporter illumination results in fluorescence resonance enrgy transfer (FRET) to the proxminal quencher without the emission of fluorescence. When the probe hybridizes tot he target DNA druing PCR, the reporter is freed from the probe, and thus from the quencher, by the 5′ exonuclease activity of Taq polymerase. Each PCR cycel will generate a free fluorescent reporter for each template amplified.

Tth DNA polymerase: Thermus thermophilus DNA polymerase has both a DNA polymerase activity as well as a reverse transcriptase activity. Thus, a single tube reaction can be carried out where the switch between reverse transcriptase adn DNA polymerase activity is modulated by the manganese concentration.

DNA Sequencing, generally

In one DNA sequencing method, four nucleotides are added stepwise to the template hybridized to a primer. The PPi released in the DNA polymerase-catalyzed reaction is detected by the ATP sulfurylase and luciferase in a coupled reaction. The added nucleotides are continously degraded by nucleotide-degrading enzyme. Afte the first added nucleotide has been degraded, the enxt nucleotide can be added. As this procedure is reepated, longer stretches of the template sequence are deduced. (Ronaghi, “A sequencing method based on real-time pyrophosphate” Science 17, Jul 1998, pp. 363-365). 

Analysis of Amplification Products

After target amplification, the simple or conventional version of product detection is use of agarose gel electrophoresis after ethidium bromide staining. Several other techniques have been developed not only to visualize the products, but to enhance both the sensitivity and specificity of amplification techniques as well.

Direct Sequencing: offers direct, rpaid and accurate anlysis of amplificaiton products.

Disadvantages of PCR

PCR is not without its disadvantages. For optimal performance, pure and undegraded nucleic acids (templates ) are required. There are a number of commercially available procedures for preparing nucleic acid for PCR. (Qiagen, Inc. (Chatsworth, CA) has one procedure where nucleic acid protein complexes are lysed and disassociated with a chatropic agent. Nucleic acid is then preferentially absorbed to silica under hihg sale conditions, contaiminatns removed by washes and DNA preferentially eluted with a low salt buffer.

For RNA targets (i.e, viruses) one of the  most effective methods for preparing RNA for amplification by reverse transcriptase (RT)-PCR procudure is lysing the cells and dissociating protein-nucleic acid complexes by a chaotropic agents. The RNA is then extracted with acidic phenol-chloroform-isoamyl alchohol and concentrated by alchohol precipiation. There are a number of commercially available systems that apply such technology such as from Promega, Inc., (Madison, WI) and Life Technologies, Inc., Bethesda, MD).

Detection of Mutants:

Wild-type blocking PCR (WTB-PCR): using locked nucleic acid (LNA) ahs demosntrated high sensitivity and versatility in the detection of low percetnage mutant populations. By adding an LNA oligo (10-12 NT) complementary to the region of the hotspot, amplificaiton of the WT allele is inhibited, leading to experimentally dirven positive selection for mutant alleles. This is accomplished by designing the LNA oligo so that it anneals to the template strand during the primer annelaing step of PCR and melts form mutant template DNA, but not WT DNA, during extension. Because a single muncoeiide mismatch in the LnA-DNA hybrid greatnly decreases its melting empterature, only mutant template DNA is free to complete its extension. Thus, WT DNA is amplified linearly but mutant DNA is amplified expoentially. Traditional Sanger sequencing can then be performed. By replacing conventioanl PCR with LnA mediated wild-type blocking PCR (WTB-PCR) in Sanger sequencing, sensitivities of up to 0.1% mutant allele in a background of WT can be acheived. For the blocking locked nucleic acid (LNA), guidelines include that hte blocking LNA oligo should be about 10-15 bases and complementary to the WT template where mutant enrichment is desired. The blocking oligo is designed to have a melting temperature (Tm) that is 10-15 C above the extension temperature during thermocycling. The Tm can be adjsuted by adding or removing LnA bases or by substituting LNA bases for DNA. (Albitar, US 10,227,657)

Albitar, (US 10,227,657) discloses a method for screening a pateint for MYD88 mutations which includes the steps of isolating DNA from a sample obtained form a pateint suspected of having a condition associated with MYD88 mutations, performing PCR on the exgtracted DNA to produce amplified DNA while blocking amplification of wT DNA, sequencing the amplified DNA, analyzing an output to identify mutations in the sequence. In one embodiment, the amplification of WT DNA is blocked by a LNA oligo which covers amino acids Q262-1266. A single nucloetide mismatch in the LNA-DNA hybride decreases Tm by up to 30C. By designing the LNA oligo to have a Tm of 10-15 C above the termpature during etension, amplificaiton of WT DNA is clocked while allowing amplificaiton of mutant DNA. 

–With Next Generation Sequencing (NGS):

Albitar (US 10,329,605) disclsoes a method for detecting a low occurrence mutation in isoalted DNA by adding a blocking probe to reagetns druing amplificaiton of the isoalted DNA. The blocking probe is an oligo complementary to WT DNA corresponding tot eh sample. The probe spans a site of a suspected mutation within a region of interest in the isolated DNA. After amplificaiotn, fragments of the amplified DNA is sequenced using NGS. Locked nucleic acids (LNAs) are a nucleic acid analog that may be used for increasing oligo hybridization strenght and specificity. The LNA bases can be incorporated into any DNA or RNA oligo and induce a conformational change in the local helix. Thsi altered state provdies the LNA bases with stronger binding strenght for complementary sequences, great mismatch discrimination, and enhanced duplex formation. These features increase amplification success when LNA are incorproated into olgios and also increase duplex melting temperatures, which enable probes and primers to be shortened and give greater specificity. Applications for LNA include allele-specific PCR, TaqMan and Molecualr Beacon probles, real-time PCR probes, antisense oligos, microarray probes and PCR primers. In principle, adding LNA or BNA (Block Nucleic Acid) corresponding to the WT DNA to prevetn amplificaiton and to seelctively sequence mutant DNA can be done using any nucleic acid that prevents the amplification of WT, which is a necesary step in amplicon-based NGS as well as to a lesser degree in hybrid capture NGS. 

Technical Approaches toward identifying Proteins with DNA binding activities

An important class of protein-protein interactions is that of proteins interacting with specific nucleic acid sequences, such as in the promoter elements of genes. These interactions involve not only interactions between several proteins associated with transcription and repair, but also critical interactions with defined nucleotide sequences.

1. Electrophoretic Mobility Shift Assay (EMSA): This technique has been instrumental in identifying binding sites for . Here, a radioactively labeled promoter or enhancer sequence is incubated with a nuclear extract containing a DNA binding protein. That DNA-protein complex is run on one lane of a gel and the DNA alone is run on another lane. The DNA-protein complex lane will have an extra slower running band (corresponding to the DNA-protein complex) in addition to the faster running free DNA band.

The DNA-binding proteins seen in EMSA can be identified using 2-D electrophoresis coupled with mass spectrometry. T

2. Chromatin immunoprecipitation (ChIP) is the best test to insure your protein binds with your promoter. In this assay you fix and lyse your cells, shear the DNA and then use an antibody which binds to your promoter to precipitate your DNA. For example, you might use the ChIP assay to assess histone H4 acetylation at theIl4 and IFNg regulatory regions. To do this you could purify T cells from the spleen and lympho nodes of mice. Chromatin complexes are then immunoprecipitated with antiboides to acetylated histones H4(+). PCR primers specific for the Il4 promoter is then used to amplify the precipitated DNA.