PCR has developed over the years and the number of variations has grown rapidly.
Nested PCR is intended to reduce the contamination of products due to non-specific amplification. The first set of primers is designed from sequences either side of the region to be amplified. The product from this reaction is then used, either directly or after purification, in another PCR with a second internal set of primers. This is very successful, but requires more detailed knowledge of the sequences involved;Learn more
This is a method allowing the direct sequencing of genomic DNA. The DNA is cut up chemically resulting in a collection of fragments. A primer is added and will anneal to complementary fragments of interest. It is then extended to produce blunt-ended dsDNA. Linkers with a staggered and blunt end can then be added to the dsDNA. This means the DNA has two known ends, one starting with the original primer and the other with the linker sequence. Subsequent PCR allows for the amplification of the DNA of interest. This may be used in the study of methylation or other conditions which may be lost during cloning and of DNA:protein interactions;
Inverse PCR is a method used to allow PCR when only one internal sequence is known. This is especially useful in identifying flanking sequences to various genomic inserts. This involves a series of digestions and self ligation before cutting by an endonuclease, resulting in known sequences at either end of the unknown sequence;
RT-PCR (Reverse Transcription PCR) is the method used to amplify, isolate or identify a known sequence from a cell's or tissue's RNA. Essentially normal PCR is preceded by transcription with reverse transcriptase (to convert the RNA to cDNA ). In this first step the following primers can be used:
- Random primers;
- Oligo dT;
- A specific primer.
The resulting cDNA can now be used in a standard polymerase chain reaction.
To avoid contamination a one step RT-PCR can be performed. One enzyme that can be used for this is rTth, a heat stable DNA polymerase that can reverse transcribe RNA. Both steps of the reaction are carried out in the same tube. Reverse transcription is carried out at 60°C and then PCR is performed.
This method is widely used in expression mapping, determining when and where certain genes are expressed and has the advantage of containing no intron sequences:Learn more
Assembly PCR is the completely artificial synthesis of long gene products by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions and the overlapping segments serve to order the PCR fragments so that they selectively produce their final product. The ability to make complete synthetic genes and the alteration of codon usage in a gene to improve protein expression in a foreign cell are amongst the uses of this technique;
One strand of the original DNA is amplified more than the other. Uses include some types of sequencing and hybridisation probing where it is an advantage to have only one strand. A large excess of primer complementary to the required strand is used but all other conditions stay the same but the number of cycles is increased;
Quantitative PCR (qPCR) is used to rapidly measure the quantity of PCR product (preferably in real-time). It is an indirect method to quantitatively measure starting amounts of DNA, cDNA or RNA. A common use is the determination of the presence/absence of a sequence or the number of copies present. It is a technique used in the determination of viral load, the response to therapeutic agents and the characterisation of gene expression.
- Quantitative real-time PCR is often referred to as RT-PCR (Real Time PCR) - DO not confuse with reverse transcriptase PCR. A better name for this method is QRT-PCR. Fluorescent dyes and probes are used to measure the amount of amplified product produced in real time. A probe with a reporter and quencher molecule at either end is added to the reaction. It binds to the denatured DNA and as the reporter and quencher are very close to each other there is no signal produced. As the PCR proceeds the probe is broken down by the 5'-3' exonuclease activity of the Taq polymerase. The fluorescent reporter and quencher are no longer in close proximity and a signal is detected;
- Quantitative competitive PCR relies on the
addition of serial dilutions of competing DNA or the addition of an internal
Touchdown PCR is a variant of PCR that reduces non-specific primer annealing by gradually lowering the annealing temperature between cycles. Specific priming occurs at higher temperatures and the lower temperatures then allow more efficient binding;
prevents non-specific amplification in the early stages of the reaction. The reaction components are heated to 95°C before adding the polymerase. Methods to inhibit the polymerase's activity at ambient temperatures, either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step have been developed;
Rapid amplification of cDNA ends. This improves the amplification of the start and finish of cDNAs;
After transformation bacterial clones can be screened for the correct recombinant DNA products using this method. Colonies are picked with a sterile toothpick and dabbed into the PCR master mix or sterile water. Primers (and the master mix) are added and the reaction started with an extended time at 95°C;
In this case several sets of unique primer pairs are used in a single reaction to give products of varying, specific, sizes. Thus information for several genes may be gained in one reaction saving time and resources. Each primer set must work at the selected annealing temperature and the products be distinguishable in size. This protocol is used in DNA profiling, detection of foreign organisms and clinical diagnoses.