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.