DNA Sequencing BCH 446

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

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

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

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

Maxam-Gilbert sequencing - modifications

Maxam-Gilbert sequencing - summary

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

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

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

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

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

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