Tuesday, August 26, 2008

DNA Sequencing Methods

Chain Termination Method (Sanger et al 1977)




In this method sequence of single stranded DNA molecule determined.




This is done by enzymatic synthesis of complementary polynucleotide chain. These chains terminate at specific nucleotide positions.




Principle

Single stranded DNA differing in length by single nucleotide can be separated by polyacrylamine gel electrophoresis. So we can get lengths from 10 to 1500 nucleotide into series of bands.




Steps

  • Starting material is identical single stranded DNA molecule.


  • Then short oligonucleotide is annealed to each single stranded molecule at same position. These gonucleotide acts as primer.


  • Strand synthesis requires DNA polymerase , dNTPs (Deoxyribonuleotide triphosphate) as substrate.


  • The DNA synthesis doesn't continue for long because along with dNTPs small amount of ddNTPs( Dideoxyribonucleotide triphosphate – It lacks 3'-hydroxyl group need to form connection with next nucleotide) is added.


  • The polymerase can't discriminate ddNTPs and dNTPs , so when corporated is growing chain causes termination i.e. blocks next nucleotide addition.



Chain Termination Sequencing

  • If ddATP is present then it causes termination at position opposite in T of template DNA


  • But as dATP is also present
    termination may not occur at first T. It will continue until ddATP is incorporated


  • So a set of new chains all of different lengths ending with ddATP is produced


  • These chains is loaded into one lane


  • Similarly family generated form ddGTP, ddCTP, ddTTP is loaded into 3 adjacent lanes


  • Then electrophoresis is done.


  • Now the sequence can be directly read for position of band in gel



Production of Single Stranded Template

  • Cloning DNA in Plasmid Vector: Resulting DNA is double stranded. It is converted to single stranded by denaturation with alkali or boiling
  • Shortcoming: Difficult to prepare sample which is not contaminated with small quantities of bacterial RNA and DNA which may act as spurious primer or template.

  • Cloning DNA in Bacteriophage M13 Vector: M13 bacteriophage has single stranded DNA genome. It is converted to double stranded replicative form after infection. The advantage is that these replicative forms can be manipulated in same way as plasmid vector i.e. inserted by restriction followed by ligation. Then single stranded DNA clone can be obtained from multiplied M13 bacteriophage after infection. The disadvantage is DNA fragments inserted longer than 3kb
    suffer deletions or rearrangement.


  • PCR: There are various ways. One of them is carrying out PCR with one normal primer and one labeled with metallic bead. Labeled strand can be purified by using magnetic device.







Properties of DNA polymerase for Chain Termination Sequencing

  • High Processivity: So that it doesn't dissociate form template before incorporation chain terminating nucleotide
  • Negligible or Zero 5' 3' Exonuclease Activity: Removal of nucleotides for 5' ends of newly synthesized strands alters lengths of these strands. So, it becomes impossible to read sequence from banding pattern
  • Negligible or Zero 3'5' Exonuclease Activity: To assure polymerase does not remove chain termination nucleotide once incorporated.

The polymerase with these properties doesn't occur naturally. It is produced artificially by modifying enzyme.

  • Klenow Polymerase: It is version of E.Coli Polymerase I with 5' 3' exonuclease activity removed. It has low processivity. So, gives non-specific bands called shadow bands. Hence it is not used now.
  • Sequenase: It is modified version of DNA polymerase of T7 bacteriophage. It has high processivity and no exonuclease activity. Hence this is the enzyme which is used in chain termination reaction.