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

polymerase chain reaction The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) t ...

that is used to amplify DNA with only one known sequence. One limitation of conventional PCR is that it requires primers 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 may refer to: *Insert (advertising) *Insert (composites) *Insert (effects processing) *Insert (filmmaking) *Insert key on a computer keyboard, used to switch between insert mode and overtype mode *Insert (molecular biology) *Insert (SQL) *Fi ...

locations. For example, various

retrovirus A retrovirus is a type of virus that inserts a DNA copy of its RNA genome into the DNA of a host cell that it invades, thus changing the genome of that cell. Once inside the host cell's cytoplasm, the virus uses its own reverse transcriptase ...

es and

transposon A transposable element (TE, transposon, or jumping gene) is a nucleic acid sequence in DNA that can change its position within a genome, sometimes creating or reversing mutations and altering the cell's genetic identity and genome size. Transpo ...

s randomly integrate into

genomic DNA Genomic deoxyribonucleic acid (abbreviated as gDNA) is chromosomal DNA, in contrast to extra-chromosomal DNAs like plasmids. Most organisms have the same genomic DNA in every cell; however, only certain genes are active in each cell to allow for c ...

. 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 with DNA databases to locate the sequence which has been disrupted. The inverse PCR method involves a series of

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'' (this term is used for other procedures as well). Hartl and Jones describe it this way: ...

s and

ligation Ligation may refer to: * Ligation (molecular biology), the covalent linking of two ends of DNA or RNA molecules * In medicine, the making of a ligature (tie) * Chemical ligation, the production of peptides from amino acids * Tubal ligation, a meth ...

, 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 thermostable

DNA polymerase A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create ...

: # A target region with an internal section of known sequence and unknown flanking regions is identified # Genomic DNA is digested into fragments of a few

kilobase A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DNA ...

s by a usually low-moderate frequency (6-8 base) cutting

restriction enzyme A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...

. # Under low DNA concentrations or quick ligation conditions, self-ligation is induced to give a circular DNA product. # PCR is carried out as usual with the circular template, with primers complementary to sections of the known internal sequence pointing outwards. Finally the sequence of the sequenced PCR product is compared against sequence databases. It is used in case of chromosome crawling.

References

{{DEFAULTSORT:Inverse Polymerase Chain Reaction Laboratory techniques Molecular biology Polymerase chain reaction