Hot Start PCR - HotStart Polymerase Chain Reaction.
Hot Start PCR allows the inhibition of polymerase activity during PCR reaction preparation. By limiting polymerase activity prior to PCR cycling, Hot Start PCR reduces non-specific amplification and increases PCR product target yield. Hot Start PCR is commonly performed by using included chemical modifications, wax-barrier methods, and inhibition by a taq-directed antibody.
Inverse PCR also called IPCR, and was first described by Ochman et al. in 1988 (1).
A limitation of standard PCR is that 5' and 3' flanking regions of your DNA fragment of interest must be known. Inverse PCR allows you to conduct PCR when you only have the information of one internal sequence.
Inverse polymerase chain reaction is a variant of PCR, and is used when only one internal sequence of the target DNA is known. It is therefore very useful in identifying flanking DNA sequences of genomic inserts. Similar to other PCR methods, inverse PCR amplifies target DNA using DNA polymerase.
Inverse PCR uses standard PCR (polymerase chain reaction), however it has the primers oriented in the reverse direction of the usual orientation. The template for the reverse primers is a restriction fragment that has been ligated upon it to form a circle.
The Inverse PCR Method
The inverse PCR method includes a series of digestions and self-ligations with the DNA being cut by a restriction endonuclease. This cut results in a known sequence at either end of unknown sequences.
Inverse PCR Steps
1)Target DNA is lightly cut into smaller fragments of several kilobases by restriction endonuclease digestion.
2) Self-ligation is induced under low concentrations causing the phosphate backbone to reform. This gives a circular DNA ligation product.
3) Target DNA is then restriction digested with a known endonuclease. This generates a cut within the known internal sequence generating a linear product with known terminal sequences. This can now be used for PCR (polymerase chain reaction).
4) Standard PCR is conducted with primers complementary to the now known internal sequences.
Inverse PCR functions to clone sequences flanking a known sequence. Flanking DNA sequences are digested and then ligated to generate circular DNA.
PCR primers pointing away from the known sequences are then employed to amplify the flanking sequences.
Applications of Inverse PCR
Inverse PCR has numerous applications in molecular biology including the amplification and identification of sequences flanking transposable elements, and the identification of genomic inserts.
Definition: Nested PCR is a variation of the polymerase chain reaction (PCR), in that two pairs (instead of one pair) of PCR primers are used to amplify a fragment.
The first pair of PCR primers amplify a fragment similar to a standard PCR. However, a second pair of primers called nested primers (as they lie / are nested within the first fragment) bind inside the first PCR product fragment to allow amplification of a second PCR product which is shorter than the first one.
The advantage of nested PCR is that if the wrong PCR fragment was amplified, the probability is quite low that the region would be amplified a second time by the second set of primers. Thus, Nested PCR is a very specific PCR amplification.
The Nested PCR Reaction
Nested PCR requires two sets of primers which are used to amplify a specific DNA fragment using two separate runs of PCR. The second pair of primers function to amplify a smaller specific DNA fragment located within the first PCR product.
Steps of the Nested PCR
Step One: The DNA target template is bound by the first set of primers shown in blue. The primers may bind to alternative, similar primer binding sites which give multiple products however only one of these PCR products give the intended sequence (multiple products not shown).
Step Two: PCR products from the first PCR reaction are subjected to a second PCR run however with a second new set of primers shown in red.
As these primers are NESTED within the first PCR product, they make it very unlikely that non-specifically amplified PCR product would contain binding sites for both sets of primers. This nested PCR amplification ensures that the PCR product from the second PCR amplification has little or no contamination from non-specifically amplified PCR products from alternative primer target sequences.
Reverse Transcription Polymerase Chain Reaction - RT-PCR
Reverse transcription polymerase chain reaction (RT-PCR) is based on the polymerase chain reaction (PCR). More importantly it is based on the process of reverse transcription, which reverse transcribes RNA into DNA and was initially isolated from retroviruses.
The techniques of RT-PCR allows the formation of cDNA (complementary or copy DNA) from RNA, which stores the sequence of RNA (such as messenger RNA, mRNA) in the more stable form of nucleic acid, DNA. This reverse transcription from RNA into its reverse complement DNA (cDNA) is the first step of a usually two-step process of RT-PCR. Furthermore, by copying the RNA into DNA, one can then amplify the cDNA sequence by using primers specific for the DNA sequence. This amplification is the final second major step of the two-step process of RT-PCR.
The Process of RT-PCR
The First step of RT-PCR is referred to as the "first strand reaction". In the first-strand reaction, complementary DNA also termed cDNA, is made from the messenger RNA template of interest using oligo dT (oligonucleotide poly-dTs act similar to primers and bind to the 3' polyA sequence located at the 3' UTR - untranslated region, which are present in most mRNAs), dNTPs, and an RNA-dependent DNA polymerase, reverse transcriptase, through the process of reverse transcription.
These factors are combined in a reverse transcriptase buffer for 1 hour at 37°C. After reverse transcriptase reaction is complete, and the cDNA has been synthesized, RNaseH is added (an RNA digestion enzyme) which digests the RNA away from the RNA-cDNA hybrid. After incubation with RNaseH, standard PCR or polymerase chain reaction is conducted using DNA oligo primers specific for the sequence of interest. This second step is referred to as the "second strand reaction".
Thus by adding the thermostable DNA polymerase, upstream and downstream DNA primers, the single stranded DNA becomes double stranded and is amplified, allowing the detection of even rare or low copy mRNA sequences by amplifying its complementary DNA.
Applications of RT-PCR
The exponential amplification of complementary sequence of mRNA or RNA sequences via reverse transcription polymerase chain reaction allow for a high sensitivity detection technique, where low copy number or less abundant RNA molecules can be detected. It is also used to clone mRNA sequences in the form of complementary DNA, allowing libraries of cDNA (cDNA libraries) to be created which contain all the mRNA sequences of genes expressed in a cell. Furthermore, it allows the creation of cDNA constructs which were cloned by RT-PCR and allow the expression of genes at the RNA and protein levels for further study.
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