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Recombinant DNA Technology (Continued)

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Western University
Biochemistry 2280A
Derek Mc Lachlin

Brandl Lecture 6 Notes 11/18/2012 Synthetic Oligonucleotides  Fragments of single stranded DNA with defined sequence that are made synthetically  When they are short (20-30 bases), they are often referred to as primers or oligos Nucleic Acid Hybridization  Denaturation/melting  Renaturation/annealing/hybridization  Crucial in PCR and other processes DNA Denaturation  If you have a duplex DNA, and you heat it up, it will denature into two individual strands o Can be done by heating or hydroxide Melting temperature (Tm)  Given a specific DNA at defined concentration, the temperature at which 50% of the molecules are single stranded o Characteristic of every DNA molecule o At low temperatures, all DNA molecules are double stranded and at high temperatures, all DNA molecules are single stranded o Referring to chemical quantities of molecules, not one molecule  Factors that determine the Tm – intrinsic features of the molecule itself o G:C content  The more G:C content, the higher the Tm  This is because G-C base pair has three hydrogen bonds, whereas A-T only has two hydrogen bonds so it is easier to melt A-T bonds  G-C bonds have more base stacking interaction as well, thus they have a higher melting temperature o Length  The longer the DNA molecule, the higher its melting temperature  Melting temperature is approximately 4 degrees for every G:C and 2 degrees for every A:T o Degree of complementarity  Degree of complementarity between the two nucleic acid strands  DNAs that are not perfectly complementary (not perfect matches) will anneal or hybridize  If there are too many mismatches, they will not hybridize at all, however – thus, some mismatch can be tolerated  The more mismatch, the lower the melting temperature  Factors that determine the Tm – extrinsic features o Salt concentration  The higher the salt concentration, the higher the Tm  Positively charged ions shield the repulsive interactions of the phosphate backbone o Solvents  Solvents that disrupt hydrogen bonding, disrupt base pair interactions and lower the Tm  Tend to make the DNA melt more easily  Urea is often used to disrupt hydrogen bonding in proteins  Melting is reversible o Process is called hybridization or annealing o Duplex is reformed – strands come back together again o In order to renature or anneal, it is required that the strands be complementary Polymerase Chain Reaction  Exponential amplification of any DNA from a source in which it is found a
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