New sequences of nucleotides can be generated in DNA not only by mutation, but also by the formation of new arrangements of genes by the movement of blocks of DNA. 2 types of recombination are called homologous or general recombination and site-specific recombination.

In Homologous recombination parent DNA duplexes align at regions of sequence similarity and new DNA molecules are formed by the breakage and joining of homologous segments. General recombination requires specialized proteins such as the RecA protein which catalyzes a reaction between a DNA double helix and a homologous region of single stranded DNA. The region of homology is identified before the duplex DNA target is opened up through a 3 stranded intermediate in which the DNA single strand forms transient base pairs with bases that flip out from the helix in the major groove. In most cases, a recombination intermediate called the Holliday junction is thought to form. In this junction, the 2 homologous DNA helices have paired and are held together by the reciprocal exchange of 2 of the 4 strands present, one originating from each of the helices. The structure contains 2 noncrossing (outside) strands and 2 crossing (inside) strands. 

Homologous recombination is particularly important in meosis where and endonuclease make a double strand break in one chromosome, an exonuclease degrades the 5′ ends of the helix so as to result in protruding 3′ ends which then seek out and find the homologous region of a second chromosome. Homologous recombination can lead to cases where the standard rules of genetics is violated. It is a law of genetics that each parent should make an equal genetic contribution to an offspring, which inherits one complete set of genes form the father and one from the mother. Thus a diploid cell that undergoes meiosis should produce 4 haploid cells, exactly half of the genes in these cells should be maternal and half paternal. But occasionally this is not the case due to something called gene conversion which is the result of general recombination and DNA repair. This could happen for example where a recombination event forms a joint where 2 paired DNA strands are not identical in sequence and thus have mismatched base pairs. If the mispaired nucleotides in say one of the 2 strands is recognized and removed by  DNA mismatch repair, an extra copy of the DNA sequence on the opposite strand is produced.

While general recombination can result in the exchange of alleles between chromosomes, the order of the genes on the interacting chromosomes typically remains the same. On the other hand, site specific recombination can alter gene order and also add new information. Site specific recombinatin moves specialized nucleotide sequenced called mobile genetic elements between nonhomologou sties with a genome. The movement can occur between 2 different positions in a single chromosome as well as between 2 different chromosomes. Unlike general recombination, site specific recombination is guided by recombination enzymes that recognize short, specific nucleotide sequences present in one or both of the recombining DNA molecules. For example, transposons use a specific enzyme usually encoded by the transposon called a transposase that acts on a specific DNA sequence at the end of the transposon, first disconnecting it from the flanking DNA and then inserting it into a new target DNA site. There is no requirement for homology between the ends of the element and the insertion site.

Transposons (transposable elements): 

Transposalbe elemtns, also called transposons and mobil genetic elemtns, are sequences of DNA able to move from one locaiton in the genome to another. They ove around in different ways. In some cases, the transpon is duplicated, and the dupolicated DNA moves to a new place in the genome. When this happens, the number of copies of the transpon increases. Other types of transposons are exised without duplication and insert themselves in the genome. 

The mechanism used by transposons for site specific recombination varies depending on the transposon. For example, DNA only transposons have short inverted repeat DNA sequences at their ends. DNA based or Class II transposons are mobile genetic elements that move in the host genome via a “cut-and-past” mechanism. They carry a transposase protein flanked by inverted terminal repeats (ITRs), which carry transposase binding sites. Any DNA flanked by the ITRs will be recognized by the transposase and will become enzymatically integrated into nuclear DNA. A transposase brings the 2 inverted sequences together, forming a DNA loop, introduces cuts at both end of the loop and catalyses an attack on a DNA target molecule. The break in the donor can be returned to its original state through a homologous end joining reaction or it can be simply resealed in which case the DNA sequence is altered. The vast majority of bacterial transposons are DNA only types. Retroviral like retrotransposons also move via DNA breakage and joining but RNA has a key role as a template to generate the DNA recombination substrate. Transposons have been developed as important tools for transgenesis in flies, fish, frogs, mice and rats.

Human chromosomes contain the following four types of transposable elements:

Long interspersed elemnts (LINEs): These ancient and successful elemtns are about 6k bp long, and they encode the enzyems needed for transposition. LINEs encode a reverse transcriptase enzyme that can make a cDNA copy of the transcribed LINE RNA. The result is a double-stranded DNA fragment that can reinsert into the genome rather than undergo translation into a protein. Since these elements use an RNA intermediate, they are called retrotransposons. 

Short interspersed elements (SINEs): are similar to LINEs but do not encode the transposition enzymes so they cannot move without using the transposition machinery of LINEs. Nested within the genome’s LINEs are over half a million copies of a SINE element called Alu which is 300 bp and represents 10% of the human genome. The Alu SINE can use the enzymes of the LINE of which it is a part to jump to a new chromosome location. Alu sequences can also jump into coding DNA sequences, causing mutations. 

Long Terminal Repeat (LTR): also use reverse transcriptase to create double stranded copies of itself that can integrate into the cell’s genome. 

Dead transposons: occupy about 3% of the genome. They are transposons inactivated by mutation that can no longer move. 

CRISPR Insertion or Deletion of Specific DNA sequences

In 1972, Cohen and Boyer disclosed the ability to disect DNA at specific sites in the DNA sequence. CRISPR provies a mechanisms for inserting or deleting specific DNA sequences using CRISPR-associated targeting RNA and the Cas9 RNA guided DNA endonuclease enzyme. It provides specificity for altering DNA. 

Numerous patents have been filed on non-naturally occurring CRISPR-Cas systems comprising vectors having a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with a target sequence of DNA molecule in a eukaryotic cell that ocntains the NA molecule and a second regulatory element operabe in a eukaryotic cell operably linked to a nucleotide sequence encoding a Type-II Cas9 protein. (see for example US 2014/0068797).