1. DNA Sequencing BCH 446

2. DNA sequencing Determination of nucleotide sequence
the determination of the precise sequence of nucleotides in a sample of DNA
Two similar methods:
1. Maxam and Gilbert method
2. Sanger method
They depend on the production of a mixture of oligonucleotides labeled either radioactively or fluorescein, with one common end and differing in length by a single nucleotide at the other end
This mixture of oligonucleotides is separated by high resolution electrophoresis on polyacrilamide gels and the position of the bands determined 11

3. The Maxam-Gilbert Technique
Principle - Chemical Degradation of Purines
Purines (A, G) damaged by dimethylsulfate
Formic acid modifies G &A
Methylation of base
Heat releases base
Alkali cleaves G
Dilute acid cleave A>G

4. Maxam-Gilbert Technique
Principle- Chemical Degradation of Pyrimidines
Pyrimidines (C, T) are damaged by hydrazine
Piperidine cleaves the backbone
2 M NaCl inhibits the reaction with T

5. Maxam and Gilbert Method
Chemical degradation of purified fragments (chemical degradation)
The single stranded DNA fragment to be sequenced is end-labeled by treatment with alkaline phosphatase to remove the 5’phosphate
It is then followed by reaction with P-labeled ATP in the presence of polynucleotide kinase, which attaches P labeled to the 5’terminal
The labeled DNA fragment is then divided into four aliquots, each of which is treated with a reagent which modifies a specific base
1. Aliquot A + dimethyl sulphate, which methylates guanine residue
2. Aliquot B + formic acid, which modifies adenine and guanine residues
3. Aliquot C + Hydrazine, which modifies thymine + cytosine residues
4. Aliquot D + Hydrazine + 5 mol/l NaCl, which makes the reaction specific for cytosine
The four are incubated with piperidine which cleaves the sugar phosphate backbone of DNA next to the residue that has been modified 11

6. Maxam-Gilbert sequencing - modifications

7. Maxam-Gilbert sequencing - summary

8. Advantages/disadvantages Maxam-Gilbert sequencing
Requires lots of purified DNA, and many intermediate purification steps
Relatively short readings
Automation not available (sequencers)
Remaining use for ‘footprinting’ (partial protection against DNA modification when proteins bind to specific regions, and that produce ‘holes’ in the sequence ladder)
In contrast, the Sanger sequencing methodology requires little if any DNA purification, no restriction digests, and no labeling of the DNA sequencing template

9. Original Sanger Method
Random incorporation of a dideoxynucleoside triphosphate into a growing strand of DNA
Requires DNA polymerase I
Requires a cloning vector with initial primer (M13, high yield bacteriophage, modified by adding: beta-galactosidase screening, polylinker)
Uses 32P-deoxynucleoside triphosphates

10. Sanger Method
in-vitro DNA synthesis using ‘terminators’, use of dideoxi- nucleotides that do not permit chain elongation after their integration
DNA synthesis using deoxy- and dideoxynucleotides that results in termination of synthesis at specific nucleotides
Requires a primer, DNA polymerase, a template, a mixture of nucleotides, and detection system
Incorporation of di-deoxynucleotides into growing strand terminates synthesis
Synthesized strand sizes are determined for each di-deoxynucleotide by using gel or capillary electrophoresis
Enzymatic methods

11. Dideoxynucleotide 5’ BASE PPP O CH2 O 3’ no hydroxyl group at 3’ end
prevents strand extension 12

12. The principles
Partial copies of DNA fragments made with DNA polymerase
Collection of DNA fragments that terminate with A,C,G or T using ddNTP
Separate by gel electrophoresis
Read DNA sequence 13

13. 3’ CCGTAC 5’ primer 5’ 3’ dNTP ddTTP ddCTP ddGTP ddATP GGCA GGCAT GGC
A T C G 14

16. Comparison Sanger Method Maxam Gilbert Method Enzymatic
Requires DNA synthesis
Termination of chain elongation
Maxam Gilbert Method
Chemical
Requires DNA
Requires long stretches of DNA
Breaks DNA at different nucleotides