|
|
EDITORIAL |
|
|
|
| Year : 2006 | Volume
: 12
| Issue : 1 | Page : 3 |
| |
PCR - From diagnostics to gene expression profiling
Roshan B Colah
Institute of Immunohaematology (ICMR), 13th floor, N M S Building, KEM Hospital, Parel, Mumbai - 400 012, India
Correspondence Address:
Roshan B Colah
Institute of Immunohaematology (ICMR), 13th Floor, N M S Building, KEM Hospital, Parel, Mumbai - 400 012
India
DOI: 10.4103/0971-6866.25294
How to cite this article:
Colah RB. PCR - From diagnostics to gene expression profiling. Indian J Hum Genet 2006;12:3 |
Our approach to the way we look at genes or analyse DNA completely revolutionized after Kary Mullis envisioned a means of copying DNA in a chain reaction in 1983. Subsequently, he won the Nobel Prize for inventing this powerful PCR technology which has now become a ubiquitous tool in every life science laboratory, be it for basic or applied research, for diagnostics or for teaching undergraduate students.
There are many PCR machines in the market today, starting with small personal thermal cyclers to high throughout quantitative real time PCR systems with improved applications like gene expression profiling, viral load analysis, quantitation of pathogens and SNP analysis to mention a few.
For most routine PCRs, the enzyme Taq polymerase remains the major workhorse however, several other enzymes are available for improving fidelity, specificity, yield and for amplification of longer targets. Hot start PCR systems are often useful for problematic PCR assays as well as for multiplexing. Here, an inhibitory antibody binds the DNA polymerase and only when a high temperature is reached, the antibody is released which triggers the reaction.
Inspite of all these improvements, failure of amplification still remains a problem sometimes and requires optimization of reaction conditions from time to time. In our experience, all target sequences do not amplify equally well with the same polymerase. PCR inhibitors also interfere if the nucleic acid purification is incomplete and organic solvents remain.
RNA is more difficult to work with because it has to be first reverse transcribed to cDNA. There are different approaches for reverse transcriptase PCR.
A major advance has been the development of real time PCR systems where the amount of amplicon generated is monitored and quantitated as the reaction progresses using fluorescent signals which increase in proportion to the amount of PCR product accumulated in the reaction. Different reporter molecules like TaqMan fluorogenic probes, molecular beacons or the intercalating dye SYBR Green have been used for generation of fluorescence.
In the last few years, 2 major milestones have been the development of microarrays and RNA interference (RNAi). Real time RT-PCR has also been used for validation of microarray results and for confirming the levels of gene knockdown in siRNA experiments.
In this issue of the journal, there are two articles on the application of PCR for diagnosis of retinoblastoma and Fragile X syndrome. A cost effective and quick strategy of multiplex PCR has been described for amplification of 12 CGA codons which account for about half of the retinoblastoma suspectibility gene mutations. In the case of Fragile X syndrome, the authors have used PCR for initial screening of triplet repeat expansions for FRAXA and FRAXE mutations with a view to cut down the number of cases requiring Southern blot hybridization.
|
