09_PCR_120309 - POLYMERASECHAINREACTION...

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POLYMERASE CHAIN REACTION (PCR - in vitro DNA Amplification) LABORATORY EXERCISE #9 Rev: 07:13:51 Principle(s) of the Procedure - COMPLEMENTARY - bases in one strand match up to those of the other via H bonding - The purine adenine (A) on one strand of DNA is always paired with the pyrimidine thymine (T) on the other strand - The purine guanine (G) is always paired with the pyrimidine cytosine (C) - A- H H -T and G- H H H -C Where Does Complementarily Come Into Play (in vivo)? DNA REPLICATION SEMICONSERVATIVE ” - Each new daughter double helix will contain 1 conserved strand from parental double helix and 1 newly synthesized complementary strand Each original parental strand is used as TEMPLATE for assembly of a new complementary strand Free nucleotide bases are positioned opposite their complementary base in the parental strand [A-T : G-C] There is a 10 -9 - 10 -10 error/bp [10 -6 /gene/generation] due to PROOF-READING FUNCTIONS of both DNA POLYMERASE III and I; i.e. it’s very accurate! Aside From DNA Replication, Where Else Does Complementarily Come Into Play (in vivo)? 1
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2. TRANSCRIPTION - The synthesis of RNA that is complementary to the DNA template - mRNA bears the message that will direct protein synthesis, i.e. will be TRANSLATED into protein - rRNA molecules are components of ribosomes, the sites of protein synthesis - tRNA molecules carry amino acids to ribosomes during protein synthesis 3. TRANSLATION -The synthesis of a polypeptide chain directed by the nucleotide sequence in a mRNA molecule The Genetic Code - The language of the genetic instructions that direct protein synthesis - “Colinearity ” - For polypeptide-coding genes, the DNA base sequence corresponds to the amino acid sequence of the polypeptide There are 20 amino acids, so there must be at least 20 code words or CODONS If 2-base pair code - 4 2 = 16 possible codons If 3-base pair code - 4 3 = 64 possible codons Wobble ” occurs - - Complementarity between the 1 st and 2 nd base pairs of the codons on the mRNA is "sufficient" to establish hydrogen bonding with anticodons on the tRNAs - Mismatches between the 3 rd base pair of a codon-anticodon are "tolerated" during translation - So, the 3’-end of the mRNA codon can vary - This “sloppy” base-pairing with the 5’- end of the tRNA anticodon - - Eliminates the need for a unique tRNA for each codon - Decreases the effects of mutations in the DNA sequence Transfer RNA [ tRNA ] Carries amino acids to the site of protein synthesis “Decodes” the mRNA by “translating” it into the correct amino acids There is at least one distinct tRNA for each of the 20 amino acids Each tRNA is encoded in the DNA [as it’s own gene] and is transcribed directly into tRNA [not mRNA] tRNAs consist of 73-93 nucleotides that form extensive hairpin loops [clover leaf]; they're are further folded into an L-shaped structure with an “anticodon loop” and an “acceptor stem
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This note was uploaded on 03/09/2010 for the course BIOL 371 taught by Professor Eugenemuller during the Fall '09 term at Framingham State College.

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09_PCR_120309 - POLYMERASECHAINREACTION...

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