DNA Sequencing
I.
INTRODUCTION
A. We discussed the Sanger's dideoxy chain termination method of DNA sequencing
1. This method was devised by Sanger and used dideoxynucleotides to terminate
chain elongation during DNA synthesis
B. Purpose
1. Use sequence to deduce amino acid sequence of proteins
2. Find restriction sites
3. Find introns
a) Compare genomic DNA to cDNA
4. Find DNA structures
a) Inverted repeats that might form hair-pin loops or cruxiform structures
5. Comparison of genes
a) Used to determine evolutionary relationships
6. Find variations between alleles
II.PROCEDURE
A. Obtaining single-stranded DNA
1. If double stranded DNA is used you would end up sequencing both strands
which could lead to confusion
2. To obtain single-stranded DNA, the DNA to be sequenced is cloned into a singlestranded DNA virus
a) Most commonly used single-stranded DNA virus is M13
B. Four reaction mixtures
1. DNA template
a) Provides the bases to be sequenced
2. Primer
a) An oligonucleotide provides a 3'-OH group to which DNA polymerase can
add nucleotides
3. DNA polymerase
a) Use just the Klenow fragment
(1) Lacking 3' to 5' exonuclease activity so that it will not remove
dideoxynucleotides
4. All four dNTPs
a) To be incorporated into newly synthesized DNA
5. 2',3' dideoxynucleoside triphosphates
a) These lack a 3' hydroxyl group, so when incorporated polymerization stops
b) Each of the four reaction mixtures will have a different dideoxynucleotide
(ddGTP, ddATP, ddCTP, or ddTTP)
C. Electrophoresis
1. Denature the DNA before electrophoresis
2. Each reaction mixture will be electrophoresed in a separate lane through a
polyacrylamide gel
a) Polyacrylamide is used as it has higher resolving power than agarose
(1) It can resolve DNA fragments differing in only one base length
3. Place X-ray film over gel
D. Reading the gel
1. The four separate reactions were separated within four separate lanes by
electrophoresis
2. Labeled bands will appear at each location where the dideoxynucleotide brought
that particular elongation reactions to a halt
3. Since DNA is always synthesized in the 5' to 3' direction, the fragments at the
bottom (smallest) of the gel represents the 5' end and the larger pieces at the
top represent the 3' end
4. Thus, one can read the sequence directly e.g. from the bottom to top