Biotechnology Processes DNA Sequencing Sanger dideoxy method 4

Biotechnology Processes: DNA Sequencing

Sanger dideoxy method: 4 a. k. a. Chain Termination Technique 4 How the scientists get the ATCG sequence for the DNA strand!

Animation

Step 1: Create 4 solutions 4 Each solution contains several single stranded DNA strands created using a PCR. 4 Each solution contains DNA polymerase and a primer 4 Each solution contains high concentrations of all of the 4 nucleotides, A, C, G, and T (in deoxynucleoside triphosphate form).

Step 1 Continued 4 Each solution contains 1 of the 4 nitrogen base pairs (either A, G, T or C) that has been altered to be a radioactively labelled dideoxy- nucleosides triphosphate

Dideoxynucleoside Triphosphates 4 A nucleotide in which a second oxygen has been removed from the ribose sugar 4 1 removal of oxygen gives Deoxyribose, 2 removals of oxygen gives Dideoxyribose! 4 Dideoxynucleotides are named dd. ATP, dd. CTP, dd. GTP and dd. TTP

Step 2: Replication 4 Allow the DNA to replicate, starting from the primer, forming complimentary strands 4 Since solution contains regular deoxynucleosides as they are incorporated the chain elongation continues, BUT 4 Since -OH is missing from dideoxy-nucleosides they end transcription (nothing to bind to) 4 Copied fragments end up being different lengths, depending on where the dd-nucleosides were randomly added!

Step 3: Creating an Autoradiogram 4 The DNA fragments are then loaded onto a polyacrylamide gel tray. 4 Each container in one of 4 runs. – Each of the radioactive forms of the nitrogen base are placed in a different well. 4 Placed against X-ray form.

4 Sequence of DNA read from autoradiogram. – read from positive end to negative (bottom to top) – Gives a 3' to 5' COMPLIMENTARY strand – To get original strand, must find the complimentary bases to the decoded strand reverse order, to get in 5' to 3' direction. . .