Polymerase Chain Reaction & its applications. Dr.Shubha Gopal Associate Professor & Chairperson Department of Studies in Microbiology University of Mysore Manasagangotri Mysore – 570 006. 15.12.2011 UGC Academic Staff College.
Associate Professor & Chairperson
Department of Studies in Microbiology
University of Mysore
Mysore – 570 006
UGC Academic Staff College
The Polymerase Chain Reaction (PCR) was not a discovery, but rather an invention
from Thermus aquaticus (1988)
Yellow stone National Park hot springs
PCRHow are the functions of replication achieved during PCR ???
. N/A as fragments are short
. Taq Polymerase
. Primers added to the reaction mix
Theoretical yield = 2n
ie. cycle 1 = 2, cycle 2 = 4, cycle 3 = 8, etc
eg. if you start with 100 copies after 30 cycles you will have 107, 374, 182, 400 copies
Long PCR: Used to amplify DNA over the entire length up to 25kb of genomic DNA
Nested PCR: Involves two consecutive PCR reactions of 25 cycles. The first PCR uses primers external to the sequence of interest. The second PCR uses the product of the first PCR in conjunction with one or more nested primers to amplify the sequence within the region flanked by the initial set of primers.
Inverse PCR: Used to amplify DNA of unknown sequence that is adjacent to known
Quantitative PCR: Product amplification w r t time, which is compared with a standard DNA.
Hot start PCR: Used to optimize the yield of the desired amplified product in PCR and simultaneously to suppress nonspecific amplification.
Colony PCR- the screening of bacterial (E.Coli) or yeast clones for correct ligation or plasmid products.
Pick a bacterial colony with an autoclaved toothpick, swirl it into 25 μl of TE autoclaved dH2O in an microfuge tube.
Heat the mix in a boiling water bath (90-100C) for 2 minutes
Spin sample for 2 minutes high speed in centrifuge.
Transfer 20 μl of the supernatant into a new microfuge tube
Take 1-2 μl of the supernatant as template in a 25 μl PCR standard PCR reaction.
Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production at each PCR cycle (in real time) as opposed to the endpoint detection
Traditional PCR has advanced from detection at the end-point of the reaction to detection while the reaction is occurring(Real-Time).
* amplification can be monitored real-time
* no post-PCR processing of products
(high throughput, low contamination risk)
* ultra-rapid cycling (30 minutes to 2 hours)
* wider dynamic range of up to 1010-fold
* requirement of 1000-fold less RNA than conventional assays
(6 picogram = one diploid genome equivalent)
* detection is capable down to a two-fold change
* confirmation of specific amplification by melting curve analysis
* most specific, sensitive and reproducible
* not much more expensive than conventional PCR
(except equipment cost)
* Not ideal for multiplexing
* setting up requires high technical skill and support
* high equipment cost
* intra- and inter-assay variation
* RNA liability
* DNA contamination (in mRNA analysis)