Sanger sequencing

Sanger sequencing

1. SANGER SEQUENCING Prepared by NAHIMA ANHUM Msc MICROBIOLOGY 2 nd semester SUBMITTED TO DR.WAQAS UNIVERSITY OF HARIPUR

2. DNA sequencing DNA sequencing  is the process of determining the precise order of nucleotides within a  DNA  molecule  To analyze gene structure and its relation to gene expression as well as protein conformation

3. Purpose of DNA sequencing  can compare genes or specific sequences to find out differences and similarities  classify organism, make a disease diagnosis Through the DNA sequencing would be able to know where exactly into genome or a gene is the mutation

4. SANGER SEQUENCE it was the most widely used sequencing method for approximately 25 years Developed by Frederick Sanger and colleagues in 1977

5. CONTINUE….  Also termed as Chain Termination or Dideoxy method method of determining the location of a specific place on a DNA fragment, based on where synthesis of a new DNA chain stops

6. Principle Utililizes 2',3'-dideoxynucleotide triphosphate ( ddNTPs )  Are different from dNTPs at the 3’carbon

7. dd NTPs Specially designed nucleotides Have a hydrogen atom attached to the 3' carbon rather than an OH group

8. dd NTPs They terminate DNA chain elongation as Cannot form a phosphodiester bond with the next deoxynucleotide Each ddNTP has label for different color fluorescence

9. THE METHODOLOGY  It depends on the fact that:  a) Synthesis of a double-stranded DNA segment from a single strand of DNA will be initiated in the presence of DNA polymerase, and;  b) DNA synthesis will stop if the incorporated base is in the form dideoxynucleotide instead of deoxynucleotide . 

10. CONTINUE…. Elongation of strand Random addition of dNTPs and ddNTPs When ddNTP added strand extension terminates Being random - all possible lengths of the DNA fragments are generated having a terminal fluorescence labeled ddNTP Product of this reaction is subjected to gel electrophoresis

11. CONTINUE… If ddNTP is added early the fragment will be short If ddNTP is added late, fragments will be longer Shorter fragments have faster mobility 

12. SANGER TECHNIQUE dideoxynucleoadenoside ( ddATP ) in a mixture that also contains deoxynucleoadenoside ( dATP ), as well as the other three deoxynucleotides , the synthesize of the double chain will stop when the ddATP molecule is incorporated instead of the dATP molecule.   some of the synthesizing DNA will be stopped at every point that adenosine is required.

13. CONTINUE…  The Sanger Technique uses the dideoxynucleotide for all four of the required nucleotides  There are four batches of reagents, one devoted to each nucleotide.  The same single-stranded DNA molecule is incubated in each batch with one of the nucelotides provided in the dideoxy form as well as its normal deoxy form

14. CONTINUE…. Among the four batches, synthesis has been arrested at every site in the DNA fragment You keep the batches separate and run gel electrophoresis on all four batches.

15. CONTINUE… Because the length of the migration in the electrophoresis fields depends on the size of molecule, the DNA fragments should distribute themselves in linear fashion according to size After reaction the electrophoresis was run in four different lanes and the matching was done using radiographic reading of the lanes