PCR (Polymerise Chain Reaction):
PCR an also be used to amplify a target RNA sequence. This procedure is called reverse transcriptase PCR (RT-PCR). The RNA sequence is first converted into a double-stranded nucleic acid (cDNA) sequence using a reverse transcriptase enzyme borrowed from a retrovirus. The cDNA sequence can then be amplified using the same PCR cycles already described.
RT-PCR is used for the detection of RNA viruses, such as HIV and hepatitis C, and messenger RNA (mRNA) of large DNA translocations (> 300-500 bp, such as BCR-ABL). Since RNA is not as stable as DNA, fresh samples are usually required for RNA, while paraffin-embedded archival samples can be used for DNA.
In addition to PCR, the most widely used nucleic acid amplification device, isothermal amplification techniques based on the enzymes required for the cellular process of DNA/RNA synthesis have also been developed and are available for diagnostic and scientific investigations, such as
Transcription-Mediated Amplification (TMA), Nucleic Acid Sequence Based Amplification (NASBA), RNA technology signal-mediated amplification (SMART), strand displacement amplification (SDA), rolling circle amplification (RCA), induced loop isothermal amplification (LAMP), isothermal multiple displacement amplification (IMDA), helicase-dependent amplification (HDA), single primer isothermal amplification (SPIA), and helicase-dependent circular amplification (cHDA).
The recent development of "real-time" PCR (Q-PCR) has brought great advantages to traditional PCR. As the name implies, this technique allows real-time quantification of the PCR product following each of the 40 cycles of amplification. The Q-PCR, which is a computerized device, measures after each cycle the amount of fluorescence emitted by a dye intercalated in the double helix DNA product; the amount of fluorescence is relative to the quantity of duplicates of the enhancement target.
When a certain critical copy number is reached, the amount of fluorescence increases exponentially. Q-PCR thus offers a great quantitative advantage. In addition, it is less contaminated since the entire process of amplification and quantification of the original target DNA for each sample is performed in a single sealed tube.
All this amplification of different products have many advantages like time saving, precise results, more specific to a person... But it main disadvantage is that it is expensive