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Lecture 34: "Cloning, Transformation & DNA Sequencing"

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Biochemistry 2280A
Mel Usselman

Biochemistry Lecture No. 34: Cloning, Transformation & DNA Sequencing th Tuesday November 27 , 2012 Restriction Enzymes (Cont’d): -KPN-1 is another class of restriction enzymes that binds to the palindromic sequence 5’ GGTACC 3’ & 3’ CCATGG 5’ (cutting between the cytosines) and leaves sticky ends with 3’ overhangs. SSP-1 is yet another class of restriction enzymes that binds to the palindromic sequence 5’ AATATT 3’ & 3’ TTATAA 5’ (cutting directly in the middle) and leaves blunt ends. Therefore we can see that there are in fact three classes of restriction enzymes observable, those that leave: 5’ overhangs, 3’ overhangs and blunt ends. 3’ 5’ 3’ 5’ 5’ 3’ 5’ G G T A C C A A T A T T 3’ C C A T G G 3’ T T A T A A 3’ 5’ 5’ 5’ 3’ 5’ 3’ KPN-1 cutting sequence SSP-1 cutting sequence -Some restriction enzymes cut identical sequences, but leave different overhanging ends. This is observed in BAM-H1 (5’ overhanging ends) and NLA-4 (blunt ends). BGL-2 and BAM-H1 cut different sequences, but leave the same overhangs. 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ G G A T C C G G A T C C 3’ C C T A G G 5’ 3’ C C T A G G 5’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ BAM-H1 cutting sequence NLA-4 cutting sequence 3’ 3’ 5’ 5’ 3’ BGL-2 cutting sequence Q) What is the probability that any random 4 base pair sequence is a Hae-3 site (GGCC)? 1 in 246 or 4 (1/4) . Given that the probability of any 6 base pair sequence being a NC-2 site is about 1 in 1000, how many of these sites are in the human genome? If there is 1 NC-2 site every 1000 base pairs, a genome of 3 billion basepairs will contain approximately 3,000,000 sites. -Like any other enzyme Each restriction enzyme has preferred conditions in which it functions. These include: temperature, pH and salt concentration. Like any other enzyme they have 3D conformations that are required for function and may be more or less stable. Obtaining Restriction Sites On YFG: -This is done through PCR, where from the starting mRNA, is cDNA synthesized. In essence you introduce restriction sites on YFG by having the restriction cut sites on the PCR primers. DNA Ligase: -DNA ligase is an enzyme that will reseal compatible sticky ends and much less efficiently blunt ends. It is important to understand that compatibility is key (anneallable sequences) for ligase to function and that it requires an energy source to function (ATP is most commonly used). Sticky ends will anneal when leftover that ligase then later seals up (afterwards you get a fully-bonded DNA molecule). Note that DNA has to have 5’ ends on the phosphates in order for this process to work. Steps In Cloning: -The first step of the cloning process is to digest both the plasmid and YFG with a compatible restriction enzyme or one that will give compatible overhanging ends (blunt ends don’t work quite as well), resulting in YFG and the plasmid having sticking ends. In the second step, the restriction enzyme has to be inactivated since it will interfere with later steps in cloning. Step 3 is to incubate the plasmid and then insert YFG together in the presence of DNA ligase and ATP. The sticky ends of the DNA mol
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