This document discusses different methods of DNA sequencing, including using the M13 bacteriophage and primer walking. Sequencing with M13 bacteriophage involves infecting E. coli with the rod-shaped M13 virus, which contains single-stranded DNA, and extracting its replicated double-stranded DNA. Primer walking is used to sequence larger DNA fragments by walking from the ends using overlapping nucleotides from initial fragments or primers. Both methods have advantages like not losing bits of DNA and working for long or short sequences. Applications of DNA sequencing include forensics, medicine, and agriculture.

1. DNA SEQUENCING (BY
USING BACTERIOPHAGE
M13 & BY PRIMER
WALKING
Shivani thorat
Msc 2
Roll no: 14

2. INTRODUCTION
ADENINE GUANINE CYTOSINE THYMINE

3. DNA SEQUENCING
Determine the order
Analyse gene structure and relation

4. METHODS OF DNA SEQUENCING
• Sangar’s method
• Maxam-Gilbert method
BASIC
• ShotgunADVANCED
• Primer walking
• M13 bacteriophage
NEXT
GENERATION

5. SEQUENCING BY M13
BACTERIOPHAGE
Rod shaped
Virus M13  ssDNA
ssDNA -E.coli infection- dsDNA (replicative
form)- ssDNA secretion
No harm to bacterial cell
DNA can be easily extracted

6. BLUE-WHITE COLONY SCREENING

7. Cloning MCS (Lac Z) Mix with E.coli
Blue–white
colonies
White colonies
used
Source of
ssDNA-
Bacteriophage
Sequencing by
basic methods

9. PRIMER WALKING
Walk from the ends  Nucelotides
Larger fragments of DNA (>500 bp)
Initial fragment or Primer
~50 nucelotides from end (to see overlapping)
Requires fast and accurate analysis of
sequence

11. ADVANTAGES
• Simple
• Bits of DNA not
lost
• No restriction
map needed
• Works for long
& short DNA
DISADVANTAGES
• Slow
• Oligos not
reusable
APPLICATIONS
• cDNA
sequencing
• Targetted
sequencing

12. APPLICATIONS
DNA
SEQUENCING
FORENSICS
MEDICINE
AGRICULTURE

13. REFERENCES
• Molecular biology- David Clark & Nanette Pazdernik
• Molecular biotechnology- Bernard Glick (4th ed)
• Principles of gene manipulation- Primrose (6th ed)