15) Molecular Techniques _9744_2019

Prepared by: Mrs Selvamani Nair & Ms Eva Hor Raffles Institution (2019) 1 Genetics and Inheritance (9744) Molecular Techniques 2019 Describe the principles and procedures of the polymerase chain reaction (PCR) (including its advantages and limitations) Describe the principles and procedures of gel electrophoresis . Polymerase Chain Reaction  Amplifies DNA from a limited source of DNA so that there is sufficient amount for analysis. * Process: (3 steps) 1. 1. Denaturation: Double stranded DNA denatures into single stranded DNA by heating to 95C 2. as weak hydrogen bonds between complementary bases of each strand is 3. broken due to increased molecular vibrations 2. Primer annealing: Each primer anneals specifically to the 3’end of each single stranded target DNA sequence via complementary base pairing when the temperature is lowered to 64C 3. Extension: Taq polymerase synthesizes the complementary DNA strand from the free 3OH’ end of the DNA primer by catalyzing the formation of phosphodiester bonds between dNTPS when the temperature is increased to 72C * Advantages: 1. Only a minute amount of DNA is required to carry out PCR as with each round of PCR, the number of copies of target DNA is doubled. Thus the number of desired sequence increases exponentially and there will be sufficient DNA for analysis. 2. Use of thermostable (i.e. resistant to denaturation at high temperatures) Taq polymerase allows PCR to be automated so DNA can be amplified very quickly. * Limitations: 1. Taq polymerase lacks 3’ to 5’ proofreading ability. Hence errors occurring early in the PCR reaction will get compounded with each subsequent replication cycle. 2. Knowledge of sequences flanking (i.e. at the 3’ ends of) the target sequence is required in order to design appropriate primers. 3. Taq polymerase tends to ‘fall off’ the DNA template before chain extension is complete if the strand is too long. Hence there is a limit to the size of DNA fragment (~3kb) to be amplified. 4. Minute amounts of contaminant DNA can be exponentially amplified along with target DNA and affect the reliability of the results. Agarose gel electrophoresis  separate