Inverse polymerase chain reaction (Inverse PCR) is a variant of the polymerase chain reaction

Polymerase chain reaction

The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....

 that is used to amplify DNA

DNA

Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

 with only one known sequence. One limitation of conventional PCR is that it requires primers

Primer (molecular biology)

A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. They are required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA...

 complementary to both termini of the target DNA, but this method allows PCR to be carried out even if only one sequence is available from which primers may be designed.

Inverse PCR is especially useful for the determination of insert

Insert (molecular biology)

In Molecular biology, an insert is a piece of DNA that is inserted into a larger DNA vector by a recombinant DNA technique, such as ligation or recombination. This allows it to be multiplied, selected, further manipulated or expressed in a host organism....

 locations. For example, various retrovirus

Retrovirus

A retrovirus is an RNA virus that is duplicated in a host cell using the reverse transcriptase enzyme to produce DNA from its RNA genome. The DNA is then incorporated into the host's genome by an integrase enzyme. The virus thereafter replicates as part of the host cell's DNA...

es and transposon

Transposon

Transposable elements are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste". Transposition can create phenotypically significant mutations and alter the cell's...

s randomly integrate into genomic DNA. To identify the sites where they have entered, the known, "internal" viral or transposon sequences can be used to design primers that will amplify a small portion of the flanking, "external" genomic DNA. The amplified product can then be sequenced and compared

BLAST

In bioinformatics, Basic Local Alignment Search Tool, or BLAST, is an algorithm for comparing primary biological sequence information, such as the amino-acid sequences of different proteins or the nucleotides of DNA sequences...

 with DNA databases to locate the sequence which has been disrupted.

The inverse PCR method involves a series of restriction digest

Restriction digest

A restriction digest is a procedure used in molecular biology to prepare DNA for analysis or other processing. It is sometimes termed DNA fragmentation...

s and ligation

DNA ligase

In molecular biology, DNA ligase is a specific type of enzyme, a ligase, that repairs single-stranded discontinuities in double stranded DNA molecules, in simple words strands that have double-strand break . Purified DNA ligase is used in gene cloning to join DNA molecules together...

, resulting in a looped fragment that can be primed for PCR from a single section of known sequence. Then, like other polymerase chain reaction processes, the DNA is amplified by the temperature-sensitive DNA polymerase

DNA polymerase

A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....

:

1. A target region with an internal section of known sequence and unknown flanking regions is identified
2. Genomic DNA is digested into fragments of a few kilobases by a usually low-moderate frequency (6-8 base) cutting restriction enzyme
   Restriction enzyme
   A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
   
   .
3. Under low DNA concentrations, self-ligation is induced to give a circular DNA product.
4. PCR is carried out as usual, with primers complementary to sections of the known internal sequence.*

Finally the sequence is compared with the sequence available in the data base.

• Note: although the figure suggests that the circularized ligation product is digested prior to PCR, this is not the case. PCR does not require linear products and the use of another restriction enzyme to cut the known sequence could also cut within the unknown region, resulting in a failed PCR.