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2012.03.12 - Bio 1202 Lecture Review Notes.docx

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Biology 1202B
Gardiner/ Murphy

Biology Lecture Review Notes Lecture 7 DNA Technologies  Vitro – in a test tube  Vivo – in our body  Polymerase chain reaction (PCR) - used to amplify something of interest in our DNA o Denature dsDNA to ssDNA o Anneal DNA primer to it  Design a primer that is complementary and will attach 5’ to 3’ on top strand, and another for bottom strand – they are binding to different strands and have different sequences (call them primer 1 and primer 2) o Extend with dNTP (Taq polymerase) o Cycle 1:  No end point of extension (no boundaries) – just goes  You started with 1 double stranded DNA, and end up with 2 double stranded DNA in cycle 1 o Cycle 2:  Some boundaries for end points o Cycle 3:  Some definite boundaries  2/8 dsDNA defining the specific fragment of interest (i.e. primers are defining the ends)  PCR is much faster than cloning, but cloning is used when the sequence is not known o PCR – need primers, so we need to know the sequences o Cloning – do not need to know the sequence, but we do know the sequences of the vectors (plasmids)  Dideoxy (Sanger) Sequencing of a fragment in a plasmid vector or PCR fragment o Start with a single stranded fragment of DNA o Primer replaces RNA primases in DNA synthesis o New DNA synthesis occurs in the 5’ to 3’ direction o Insertion of dideoxyribonucleotides (does not have a 3’ OH, only a hydrogen) stops synthesis o dNTP and ddNTP are being added randomly  If dNTP is added, DNA synthesis (sequencing) can continue  If ddNTP is added, DNA synthesis (sequencing) is stopped o Creates fragments of various sizes
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