Vimarsh :: View topic

Posted: Mon Aug 17, 2009 3:04 pm

Before the discovery of Taq polymerase biologists used to carry out PCR without this thermostable enzyme, how was this possible?

Posted: Mon Aug 17, 2009 4:06 pm

wikipedia wrote:

A 1971 paper in the Journal of Molecular Biology by Kleppe and co-workers first described a method using an enzymatic assay to replicate a short DNA template with primers in vitro. However, this early manifestation of the basic PCR principle did not receive much attention, and the invention of the polymerase chain reaction in 1983 is generally credited to Kary Mullis.

At the core of the PCR method is the use of a suitable DNA polymerase able to withstand the high temperatures of >90 °C (>195 °F) required for separation of the two DNA strands in the DNA double helix after each replication cycle. The DNA polymerases initially employed for in vitro experiments presaging PCR were unable to withstand these high temperatures. So the early procedures for DNA replication were very inefficient, time consuming, and required large amounts of DNA polymerase and continual handling throughout the process.

The discovery in 1976 of Taq polymerase (a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus, which naturally occurs in hot (50 to 80 °C (120 to 175 °F)) environments) paved the way for dramatic improvements of the PCR method. The DNA polymerase isolated from T. aquaticus is stable at high temperatures remaining active even after DNA denaturation, thus obviating the need to add new DNA polymerase after each cycle. This allowed an automated thermocycler-based process for DNA amplification.

Posted: Tue Aug 18, 2009 8:05 am

tk tarun wrote:

Before the discovery of Taq polymerase biologists used to carry out PCR without this thermostable enzyme, how was this possible?

I think that you would benefit much if you go through the History of PCR available on the following link:

http://www.molecularstation.com/pcr/history-of-pcr/

According to this article:

Quote:

More than 30 years ago, the introduction of recombinant DNA technology as a tool for the biological sciences revolutionized the study of life. Molecular cloning allowed the study of individual genes of living organisms; however this technique was dependent on obtaining a relatively large quantity of pure DNA. This depended on the replication of the DNA of plasmids or other vectors during cell division of microorganisms (1). Researchers found it extremely laborious and difficult to obtain a specific DNA in quantity from the mass of genes present in a biological sample (2). Recombinant DNA technology made possible the first molecular analysis and prenatal diagnosis of several human diseases. Fetal DNA obtained by amniocentesis sampling could be analyzed by restriction enzyme digestion, electrophoresis, southern transfer and hybridization to a cloned gene or oligonucleotide probes (3). However, southern blotting permitted only rudimentary mapping of genes in unrelated individuals .

Posted: Sat Jul 03, 2010 2:29 pm

the need of thermostable polymerase is eliminated if you add the polymerase after the denaturation step.
as far as i know at the time PCR technique was discoverd , there were no thermocyclers where u just need to put the entire reaction mix and set the cycle conditions.
that time they had to maintain water baths at different temperatures and shift the reaction manually.
therefore the polymerase was added only when the extension step was carried out.
the need of thermostable plomerases arose for automated chain reactions. Confused