BIO282 Lecture Notes - Lecture 8: Molecular Cloning, Reverse Transcription Polymerase Chain Reaction, Cloning
PCR LECT-6
• What is PCR and how does it work?
• PCR cycling conditions and optimizations
• Applications and limitations of PCR
• Cloning of PCR products
• Different PCR techniques and their applications
o RT- PCR
o Real-Time PCR
o AP-/RAPD-PCR
The process of DNA synthesis
• By heating to 95oC or higher, dsDNA separates into two strands.
• By cooling down the mixture, the two strands hybridize again. Primers, if present, will bind
at complementary sequences.
o Two strands coming together from a hybrid (hybridization)
o Bases can only be added if there are primers added
• DNA polymerase synthesizes new DNA by extending the primers.
DNA polymerase ads new bases based on the template and will continue synthesizing
the new strand
• In this example here, two copies of DNA are generated from one molecule.
After each cycle the amount of DNA get doubled
• 2^x = number of copies (x - number of cycles)
What is PCR?
• PCR is an acronym which stands for polymerase chain reaction.
• It is basically a primer extension reaction for amplifying specific nucleic acids in vitro.
• A specific DNA sequence is amplified by repeated cycles of replication with DNA polymerase.
• Taq polymerase
What is the purpose of PCR?
To make a substantial number of copies of a specific DNA region. This is essential to have enough
starting material if you want to analyse/visualise a gene.
Good for DNA cloning (when you have small DNA)
Forensics
How does it work?
• The target sequence is identified by a specific pair of DNA primers (oligonucleotides
usually 18-24 bases in length).
o Length is required for specificity and uniqueness to binding, to prevent random
binding
• The use of a thermostable polymerase allows the dissociation of newly formed
complimentary DNA and subsequent annealing or hybridization of primers to the target
sequence with minimal loss of enzymatic activity.
https://www.youtube.com/watch?v=eEcy9k_KsDI
Document Summary
Pcr lect-6: what is pcr and how does it work, pcr cycling conditions and optimizations, applications and limitations of pcr, cloning of pcr products, different pcr techniques and their applications, rt- pcr, real-time pcr, ap-/rapd-pcr. The process of dna synthesis: by heating to 95oc or higher, dsdna separates into two strands, by cooling down the mixture, the two strands hybridize again. Primers, if present, will bind at complementary sequences: two strands coming together from a hybrid (hybridization, bases can only be added if there are primers added, dna polymerase synthesizes new dna by extending the primers. Dna polymerase ads new bases based on the template and will continue synthesizing the new strand. In this example here, two copies of dna are generated from one molecule. After each cycle the amount of dna get doubled: 2^x = number of copies (x - number of cycles) What is pcr: pcr is an acronym which stands for polymerase chain reaction.
