0.1 Basic principles of genetics  (Page 18/19)

(3) DNA polymerase is added along with the four nucleotide precursors (dATP, dGTP, dCTP, and dTTP). The mixture is then divided into four separate reactions and to each reaction a small quantity different dideoxy nucleotide precursor is added. Dideoxy nucleotide precursors are abbreviated ddATP, ddGTP, ddCTP, and ddTTP.

(4) The polymerase reactions are allowed to proceed and, using one of a variety of methods, radiolabel is incorporated into the newly synthesized DNA.

(5) After the DNA polymerase reactions are complete, the samples are melted and run on

a gel system that allows DNA strands of different lengths to be resolved. The DNA sequence can be read from the gel by noting the positions of the radiolabeled fragments. The crucial element of the sequencing reactions is the added dideoxynuclotides. These molecules are identical to the normal nucleotide precursors in all respects except that they lack a hydroxyl group at their 3’ position (3’ OH).

A part of the final gel will look like this:

(Note that larger molecules migrate more slowly to the cathode on these gels)?

The deduced DNA sequence obtained from this gel is: 5’ GGATCCTATC 3’?

Polymerase chain reaction

(1) A crude preparation of chromosomal DNA is extracted from the bacterial strain of interest.

(2) Two short oligo nucleotide primers (each about 18 bases long) are added to the DNA.

The primers are designed from the known genomic sequence to be complimentary to opposite strands of DNA and to flank the chromosomal segment of interest.

(3) The double stranded DNA is melted by heating to 100C and then the mixture is cooled to allow the primers to anneal to the template DNA.

(4) DNA polymerase and the four nucleotide precursors are added, and the reaction is incubated at 370C for a period of time to allow a copy of the segment to be synthesized.