Unit 3-2_4-1Students

Unit 3-2_4-1Students - Unit 3-2 DNA Sequencing PCR RT PCR...

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Unformatted text preview: Unit 3-2 DNA Sequencing PCR RT PCR Blotting E c o R I E c o R I Vector 300 bp Insert 1 Kb Restriction Mapping: Example Can we determine the orientation of insert? EcoRI EcoRI MW ucut BamHI PstI BamHI + PstI 1 Kb 800 600 400 200 Wells 400 600 BamHI SINGLE EcoRI EcoRI MW ucut BamHI PstI BamHI + PstI 1 Kb 800 600 400 200 Wells 400 600 BamHI SINGLE DOUBLE 100 500 400 DOUBLE 100 500 400 PstI EcoRI EcoRI MW ucut BamHI PstI BamHI + PstI 1 Kb 800 600 400 200 Wells 100 900 PstI SINGLE EcoRI EcoRI MW ucut BamHI PstI BamHI + PstI 1 Kb 800 600 400 200 Wells 100 900 PstI SINGLE DOUBLE BamHI 400 500 EcoRI EcoRI 400 600 BamHI SINGLE DOUBLE 100 500 400 DOUBLE 100 500 400 PstI EcoRI EcoRI 100 900 PstI SINGLE DOUBLE BamHI 400 500 CANNOT Determine Orientation! Insert Vector EcoRI EcoRI Orientation of the insert can only be determined if we cut the fragment out of the plasmid with two different enzymes DNA Sequencing Technology based on DNA polymerases: Two methods: 1. Maxam-Gilbert (chemical cleavage) 2. Sanger (dideoxy chain-termination using DNA polymerase) Nobel prize, 1980 DNA polymerase require a free 3 OH What provides this free 3OH group in DNA Replication? Di deoxynucleotide triphosphate (2', 3') Terminates the growing chain during DNA synthesis 5 4 3 2 1 Template (single stranded) Primer (at known position) DNA polymerase 4 dNTPs 1 of 4 ddNTPs (low concentration) Most of the time a dNTP will be incorporated, When a ddNTP is incorporated, chain will terminate DNA Sequencing Tube 1 ddATP Tube 2 ddGTP Tube 3 ddTTP Tube 4 ddCTP A typical sequencing reaction (the old-fashioned way) Template Primer 4 dNTPs (1 radiolabeled) DNA pol All synthesized DNA will be labeled. Each tube will contain a range of different length fragments that terminate at the same base DNA Sequencing Run on polyacrylamide gel, expose to x-ray film Gel Electrophoresis Electro refers to electricity Phoresis is from the Greek to be carried Used to separate DNA, RNA or proteins according to mass Agarose Polyacrylamide Gel electrophoresis Agarose or polyacrylamide gel Well Fractionation based on molecular weight Gel electrophoresis Agarose or polyacrylamide gel Well Fractionation based on molecular weight Well Band Large Small Sequencing gel (radioactive DNA) A C G T 5'AAAAAACGGACCGGGT etc....
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Unit 3-2_4-1Students - Unit 3-2 DNA Sequencing PCR RT PCR...

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