PRESENTED BYMARIAM RAZIBS medical technology 6th semester

DNA SEQUENCING

DNA

• Deoxyribonucleic acid (DNA) is a nucleic acid that functions include

Storage of genetic information

Self-duplication & inheritance.

Expression of the genetic message.

• DNA’s major function is to code for proteins.

• Information is encoded in the order of the nitrogenous bases.

Watson & Crick Model • DNA is composed of 2 chains of nucleotides that

form a double helix shape.

• The two strands are antiparallel.

• The backbone of the DNA molecule is composed of alternating phosphate groups and sugars.

• The complimentary nitrogenous bases form hydrogen bonds between the strands.

• A is complimentary to T and G is complimentary to C.

DNA SEQUENCING

Determining the order of bases in a section of DNA

PURPOSE

• Deciphering “code of life”

• Detecting mutations

• Typing microorganisms

• Identifying human halotypes

• Designating polymorphisms

DNA SEQUENCING METHODS

• Historically there are two main methods of DNA sequencing: 1.Maxam and Gilbert method

2.Sanger method

Modern sequencing equipment uses the principles of the Sanger technique.

MAXAM & GILBERT METHOD

• A. M. Maxam and W.Gilbert-1977

• The sequence of a double-stranded or single-stranded DNA molecule is determined by treatment with chemicals that cut the molecule at specific nucleotide positions.

PRINCIPLE :

Chemical degradation

Reaction in two stages:• Chemical modification of the bases• Modified base is removed from its sugar,

pyperidin cleaves phosphodiester bonds 5’ and 3’ and base is released

SANGER METHOD

• Most common approach used to sequencing DNA.

• Invented by Frederick Sanger - 1977

• Nobel prize - 1980

• Also termed as chain termination or dideoxy method

SANGER METHOD TERMED AS CHAIN TERMINATION METHOD

This method uses dideoxynucleotide triphosphates(ddNTPs) chain terminators : which have an H on the 3’ carbon of the ribose sugar

instead of the normal OH found in deoxynucleotide triphosphates (dNTPs).

Therefore in a synthesis reaction, if a dideoxynucleotide

isadded instead of the normal deoxynucleotide, the

synthesisstops at that point because the 3’OH necessary for theaddition of the next nucleotide is absent.

DEOXY VERSUS DIDEOXY

PRINCIPLE :

The sequence of a single-stranded DNA molecule is determined by enzymatic

synthesis of complementary polynucleotide chains.

These chains terminating at specific nucleotide positions.

Separate by gel electrophoresisRead DNA sequence

REQIREMENTS

DNA sequencing is performed in four separate tubes, each containing         i.   Single stranded DNA to be sequenced          ii.  DNA polymerase          iii.  Primers          iv.  The four dNTPs (dATP, dCTP, dTTP and

dGTP)          v.   Small amount of one of the four ddNTPs (ddATP or ddCTP or ddTTP or ddGTP)

Either the primers or the dNTPs are radiolabeled with 32P

HOW DOES THE DNA TEMPLATE OBTAINED?

• The DNA can be cloned in a plasmid vector.

• The DNA can be cloned in a bacteriophage M13 vector.

• PCR can be used to generate single-stranded DNA.

COMPARISON

Sanger Method Maxam Gilbert Method

Enzymatic Chemical

Requires DNA synthesis

Requires DNA

Termination of chain elongation

Breaks DNA at different nucleotides

Automation Automation is not available

Single-stranded DNA.

Double-stranded or single- stranded DNA

Next Generation Technologies:

Solexa : Based whole genome sequencing

SOLiD : Ligation and detection454 : PyrosequencingHelicos : Single molecule sequencing

THANK YOU