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Lecture 6

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Western University
Biochemistry 2280A
Christopher Brandl

Polymerase Chain Reaction (PCR) ● Exponential amplification of any DNAfrom a source in which it is found as little as once Required reagents for PCR 1. Template DNA 2. 2 oligonucleotide primers which flank TFG 3. dNTPs 4. DNApolymerase Steps ● 1. Head separate strands; denaturation (90 C) ● 2. Hybridization of Primers (™ 60 degrees celsius); dNTPs are here too, don’t mind heating ● 3. In presence of dNTP, extend primers of both strands (72 C) ● One cycle gets you two DNAmolecules from one ● PCR is repeated, two molecules denature, anneal primers + extend; after cycle 2 → 4 molecules ● 3rd cycle → 8 molecules; start to see molecules that begin and end with primer sequences ● # of copies after n cycles is 2^n; 2^30 ~ 1 billion Two Key TechnicalAdvantages ● Discovery of thermostable DNApolymerase (taq polymerase; isolated from DNAof hot springs) ● Thermocyclers that oscillate between the 3 required temperatures (50C, 94C, 70C) ○ Run through 3 temperatures automatically; takes 1.5 hrs to do 30 cycles now How do we purify the PCR product? ● Gel Electrophoresis ○ DNAis homogenous; charge to mass ratio is similar, differ in length → separate DNAby length ○ Q: When placed in electric field at ph7, DNAwill migrate towards which pole? (+, - or both);ANS: (+) ■ DNAis negatively charged (phosphate backbone) → attracted to + charge Gel Electrophoresis ● DNAis sieved through a matrix of agarose being pulled by an electric field ○ has pores in it ○ put into a tank, DNAmigrates towards positive pull ● Gel is in a chamber, submerged in buffer.An electrical field is then applied ● DNa fragments are pulled through the agarose matrix and move towards the positive pole ○ DNAhas to sift through pores in matrix of agarose ● Smaller DNAfragments move faster than large fragments ○ Small DNAcan snake through pores vs large DNA Detection of the DNAin the gel ● When stained with ethidium bromide DNAfluoresces red under UV light ○ ethidium bromide is interlaced between bases ● Autoradiography ○ radioactively label the 5’ends of your DNAfragments using polynucleotide kinase and [32P] -ATP ○ expose your gel to xray How do we get YFG into e.coli? ● we insert it into a plasmid Plasmids ● Defn: Fragments of DNAthat replicate independently from the host chromosome ○ E.coli chromosome: one copy; 3 million base pairs ○ Plasmid DNA: copy # varies depending on plasmid from 1-100 copies/cell ■ doesn’t have to be circular but often circular ■ ~3000 bp ● Key Features required to make a plasmid useful for cloning ○ Origin of Replication ○ Aselectable marker (have to be able to find it): usually a gene encoding resistance to an antibiotic e.g., ampicillin ○ Asite into which YFG can be inserted How do we get YFG into the plasmid? ● Restriction Enzymes (very important) ○ Can be thought of as site specific DNAscissors
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