DNA sequencing by Sanger’s method

May 11, 2021 Sushil Humagain Bacterial genetics, Biotechnology, Molecular Biology 0

  • DNA sequencing is the process of determining the entire sequence of nucleotides of a genome.
  • The development of new techniques has made it possible to determine the gene sequences of organisms ranging in complexity from bacteria to humans.
  • Sanger’s method, also known as dideoxy or chain-termination method was developed by Frederick Sanger in 1977.
  • It reduces the DNA replicated from a template strand to be sequenced, into four sets of labeled fragments by interrupting the replication process at one of the four bases.

Principle:

  • This method follows the mechanism of DNA replication i.e. DNA synthesis by DNA polymerase.
  • The DNA to be sequenced is used as a template strand and a short primer (for the known sequences at 3’ end of the template strand), is radioactively or fluorescently labeled and annealed to it.
  • DNA polymerase uses 2’-deoxyribonucleoside triphosphates (dNTPs) as substrates. 3’-hydroxyl group of the primer reacts with the incoming (dNTP) to form a new phospho-diester bond elongating the strand.
  • The reaction mixture also contains modified substrates called 2’-,3’-dideoxynucleoside triphosphate (ddNTPs) which are analogs of dNTPs and lack the 3’-hydroxyl group on their ribose sugar.
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  • Once ddNTP is incorporated at the 3’- end of a growing polynucleotide chain, the lack of 3’-hydroxyl group prevents the addition of further nucleotides through phospho-diester bond causing the elongation to terminate.
  • ddNTPS are used in less amount as compared to more amount of dNTPs.
  • ddNTPs of respective dNTPs terminate chain at their respective sites. i. e. ddCTP, ddGTP, ddTTP and ddATP terminate at C, G, T and A sites respectively.
  • When this process is repeated separately for each of the four ddNTPs, different-sized radiolabeled or fluorescent-labeled nested fragments are obtained. The DNA strands differing in size even by only one nucleotide can be separated.
  • Polyacrylamide gel is used  for short DNA molecules (up to a few hundred nucleotides) and agarose gel is generally used for longer fragments of DNA.

Procedure:

  1. Preparation of template for replication:
    • The double-stranded DNA is denatured into two single-stranded DNA, taking one as a template DNA.
    • A DNA primer which is essential to initiate replication of template is either radiolabeled or fluorescent-labeled and bound with a single stranded cloning vector M13 flanked with the template strand at its 3’-end.
    • Many copies of such template DNA hybridized with labeled primer are prepared.
  1. Generation of four sets of nested labeled fragments:
    • Copies of each template are divided into four batches (labeled as A, T, C and G) and each batch is used for different replication reactions.
    • DNA polymerase I is added to all four batches containing copies of template.
    • Excess of dNTPs substrates (dATP, dGTP, dCTP and dTTP) are added to all the reaction mixtures in each batch.
    • Similarly, only one type of ddNTP is added to each respective batch labeled accordingly. i. e. ddATP is added to the batch labeled as A, ddTTP to T, ddCTP to C and ddGTP to G respectively.
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  1. Separation of nested DNA fragments by gel electrophoresis:
    • The solutions containing a mixture of labeled fragments from four batches are loaded into four different wells on polyacrylamide gel and then electrophoresed.
    • DNA being negatively charged, the oligonucleotides will be pulled toward the positive electrode on the opposite side of the gel.
    • The smaller a fragment is, the less friction it will experience as it moves through the gel, and the faster it will move. As a result, the oligonucleotides will be arranged from smallest to largest, reading the gel from bottom to top.
  2. Autoradiogram reading and determination of DNA sequence:
    • The autoradiogram of the gel is analyzed to determine the order of bases of complementary strand thus synthesized by DNA polymerase in 5’-3’ direction starting at a provided primer.
    • Each terminal ddNTP will correspond to a specific nucleotide in the original sequence. (i. e. the shortest fragment must terminate at the first nucleotide from the 5’ end; the second-shortest fragment must terminate at the second nucleotide from the 5’ end and so on).
    • Therefore, by reading the gel bands from smallest to largest, we can determine the 5’ to 3’ sequence of the original DNA strand.

DNA sequencing by Sanger’s method