1. Principles of DNA Sequencing Terry Kotrla, MS, MT(ASCP)BB Spring 2010

2. Background Rapid analysis of DNA sequence developed in 1970’s Two basic approaches – Organic chemical reactions with DNA bases – Enzymatic process Chemical method tedious, labor intensive Enzymatic (dideoxy) relatively fast

3. Background Autoradiographs http://tinyurl.com/2dg9pvx

4. Overview of Procedure Uses 17 base synthetic single stranded DNA hybridized to unique site. 17 base oligonucleotide serves as primer for DNA synthes. Four separate enzymatic reactions are performed, one for each nucleotide. – All 4 DNTPs are used (dATP, dGTP, dCTP, dTTP) – Concentrations adjusted so they incorporate into growing DNA strand randomly and infrequently – Once incorporated DNA synthesis is terminated – Allows determination of position of that base

5. Overview Reaction will contain millions of growing DNA strands, a “nested set” will be obtained. Each fragment terminated at different position. Nested set is complimentary to region being sequenced. Separate A, T and C reactions. Separation of radioactive products on gel. Resolves fragments by single nucleotides

6. Overview After electrophoresis gel placed in direct contact with x-ray film. Exposure allows visualization of fragments. Position on gel determines sequence (page 6)