Sanger’s method of gene sequencing
Sanger’s method of gene sequencing is also known as dideoxy chain termination method. It generates nested set of labelled fragments from a template strand of DNA to be sequenced by replicating that template strand and interrupting the replication process at one of the four bases.
Four different reaction mixtures are produced that terminates in A. T. G or C respectively.
- A DNA primer is attached by hybridization to the template strand and deoxynucleosides triphosphates (dNTPPs) are sequentially added to the primer strand by DNA polymerase.
- The primer is designed for the known sequences at 3’ end of the template strand.
- M13 sequences is generally attached to 3’ end and the primer of this M13 is made.
- The reaction mixture also contains dideoxynucleoside triphosphate (ddNTPs) along with usual dNTPs.
- If during replication ddNTPs is incorporated instead of usual dNTPs in thegrowing DNA strand then the replication stops at that nucleotide.
- The ddNTPs are analogue of dNTPs
- ddNTPs lacks hydroxyl group (-OH) at c3 of ribose sugar, so it cannot make phosphodiester bond with nest nucleotide, thus terminates the nucleotide chain
- Respective ddNTPs of dNTPs terminates chain at their respective site. For example ddATP terminates at A site. Similarly ddCTP, ddGTP and ddTTP terminates at C, G and T site respectively.
1. Template preparation:
- Copies of template strand to be sequenced must be prepared with short known sequences at 3’ end of the template strand.
- A DNA primere is essential to initiate replication of template , so primer preparation of known sequences at 3’end is always required.
- For this purpose a single stranded cloning vector M13 is flanked with template strand at 3’end which serves as binding site for primer.
2. Generation of nested set of labelled fragments:
- Copies of each template is divided into four batches and each batch is used for different replication reaction.
- Copies of standard primer and DNA polymerase I are used in all four batches.
- To synthesize fragments that terminates at A, ddATP is added to the reaction mixture on batch I along with dATP, dTTP,dCTP and dGTP, standard primer and DNA polymerase I.
- Similarly, to generate, all fragments that terminates at C, G and T, the respective ddNTPs ie ddCTP,ddGTP and ddTTP are added respectively to different reaction mixture on different batch along with usual dNTPs.
3. Electrophoresis and gel reading:
- The reaction mixture from four batches are loaded into four different well on polyacrylamide gel and electrophoresed.
- The autoradiogramof the gel is read to determine the order of bases of complementary strand to that of template strand.
- The band of shortest fragments are at the bottom of autoradiogram so that the sequences of complementary strand is read from bottom to top.