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Polymerase Chain Reaction

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Western University
Biochemistry 2280A
Derek Mc Lachlin

Brandl Lecture 7 Notes 11/22/2012 What do we use as the Template for PCR?  Q: If our goal is to express YF(human)G, genomic DNA cannot easily be used as the template? A: Yes but with great difficulty  Bacteria cannot handle introns, thus if genomic DNA is to be used, the introns would have to be eliminated  Introns are a problem in expressing genes in E. coli  The alternative is to use cDNA cDNA  cDNA is a replicate DNA copy of mRNA  c = complementary  cDNA does not contain introns  Each cDNA encodes one gene  The cDNA can be cloned or used in PCR Making cDNA  First step is to purify RNA through multiple steps o Major contaminant is DNA so it takes multiple steps to remove it o End up with pure RNA – starting point to make cDNA  Second step is to perform a hybridization or annealing step o Hybridize or anneal with a poly(T) primer o Primer is about 10 Ts in length  Third step is to synthesize DNA copy with reverse transcriptase o Reverse transcriptase – native enzyme that converts RNA into DNA for integration into the host genome  Fourth step is to degrade RNA with RNase H, which degrades RNA in an RNA-RNA duplex o Or can be digested with sodium hydroxide  Summary o Anneal primer o Make DNA copy with reverse transcriptase o Treat with alkali to degrade RNA  Q: Can one strand of cDNA be used in a PCR reaction? A: Yes, cDNA can be used in a PCR reaction What is the Source of the mRNA?  You need to identify a tissue or human cell line in which YFG is expressed  Hybridization approach is used, called Northern Blot  Northern blot – key feature is DNA-RNA hybrid o Requires that strands be complementary  Hybridization is a powerful tool to identify nucleic acid sequences of interest  Hybridization experiments are generally done after transferring the DNA or RNA to a nitrocellulose membrane o Southern blot: single stranded DNA to single stranded DNA o Northern blot: single stranded DNA to RNA  Example of a Northern Blot o Identify which tissues express YGF to the greatest level  Step 1: isolate RNA as group of tissues or cell lines  Step 2: separate the RNA on the basis of size by agarose gel electrophoresis  Positive pole is at the bottom, negative pole is at the top  Two major bands are seen in the gel – these bands represent ribosomal RNAs, which are abundant in the cell – serve as good standards for the gel  Step 3: transfer the RNA to a nitrocellulose membrane (blotting step)  Buffer travels through the sponge and through the gel, picking up the RNAs, and laying them out on the nitrocellulose paper  Procedure is slow, but 100% transfer of RNA from the gel to the nitrocellulose paper  Step 4: radioactively
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