VPB 321: Animal Biotechnology (2+1)

Maxam-Gilbert Method

MAXAM GILBERT METHOD OF SEQUENCING

• The chemical method of Maxam and Gilbert requires that the DNA fragment to be sequenced is labeled at one end, usually by adding either a non radioactive phosphate to the 5' or 3' end or a nucleotide to the 3' end. This method works for both single stranded and double stranded DNA and involves base - specific cleavages which occur in two steps.
  • The base is modified by specific chemicals such as Dimethyl sulfate (DMS), Formic acid and Hydrazine. The specific base modifications reactions use DMS to methylate at G base. Formic acid will attack purines A and G. Hydrazine is used to hydrolyse at pyrimidines (C+T) but high salt inhibits the T reaction. Thus four lanes on the sequencing gel (G, A+G, C+T and C) allow the sequences to be determined. This method is useful for sequencing genomic DNA without cloning.
  • After the base modification, piperidine is used to cleave the sugar phosphate back bone of the DNA.