Sanger sequencing

SANGER SEQUENCINGPrepared by

NAHIMA ANHUM Msc MICROBIOLOGY

2nd semester SUBMITTED TO

DR.WAQASUNIVERSITY OF HARIPUR

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

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

SANGER SEQUENCE it was the most widely used

sequencing method for approximately 25 years

Developed by Frederick Sanger and colleagues in 1977

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

Principle Utililizes 2',3'-dideoxynucleotide

triphosphate (ddNTPs)

 Are different from dNTPs at the 3’carbon

dd NTPs Specially designed nucleotides

Have a hydrogen atom attached to the 3' carbon rather than an OH group

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

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. 

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

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 

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.

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

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.

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