Inverse polymerase chain reaction (Inverse PCR) is a variant of the polymerase chain reaction 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 locations. For example, various retroviruses and transposons 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 with DNA databases to locate the sequence which has been disrupted.
The inverse PCR method involves a series of restriction digests and ligation, 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:
- 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 kilobases by a usually low-moderate frequency (6-8 base) cutting restriction enzyme.
- Under low DNA concentrations, self-ligation is induced to give a circular DNA product.
- 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.
... This reaction proceeds efficiently when this solution is incubated at room temperature with required salt ... are used for cloning fragments amplified by either Taq or Pfu polymerase as Taq polymerase (unlike Pfu) leaves an extra "A" nucleotide at the 3'end during ... The insert is created by PCR using Taq DNA polymerase, a polymerase that lacks 3' to 5' proofreading activity and with a high probability adds a single, 3'-adenine overhang to each end of the PCR product ...
... is an antibiotic which works by blocking nucleic acid chain elongation by binding to the polymerase, thus stopping RNA polymerase activity inside a cell ... Specifically, it inhibits the assembly of the RNA polymerase II transcription complex and DNA polymerase III transcription ...
... A polymerase is an enzyme whose central biological function is the synthesis of polymers of nucleic acids ... DNA polymerase and RNA polymerase are used to assemble DNA and RNA molecules, respectively, generally by copying a DNA or RNA template strand using base-pairing interactions ... the generation of other biopolymers are not also referred to as polymerases ...
... The T7 DNA polymerase of the T7 bacteriophage is a DNA-dependent DNA polymerase responsible for the fast rate of T7 phage DNA replication in vivo ... The polymerase consists of a 11 complex of the viral T7 gene 5 protein (80kDA) and the E ... This polymerase is unique due to its considerable processivity, or ability to stay on DNA for a greater than average number of base pairs ...
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“Yet time and space are but inverse measures of the force of the soul. The spirit sports with time.”
—Ralph Waldo Emerson (18031882)