DNA SEQUENCING

BY MONA KABYEMELA
110
SUMY STATE UNIVERSITY
 INTRODUCTION

 DNA sequencing is a technique used to determine the precise order of the four nucleotide
bases, adenine, guanine, cytosine, and thymine which make up a strand of DNA.
 For understanding the structure and function of a gene, It is very important to study its
nucleotide sequence.
HISTORY OF DNA SEQUENCING
 METHODS OF DNA SEQUENCING
Basic methods
• Maxam-Gilbert sequencing
• Chain termination (Sanger’s
method) method
Advanced methods Next-generation methods
• Short Gun Sequencing • Massively parallel signature
• Bridge PCR Sequencing sequencing
• Polony sequence
• Pyrosequencing
• Illumina sequencing
• Solid sequencing
SANGERS METHOD OF DNA
SEQUENCING
The most common approach is used to sequence DNA.
 Invented by Frederick Sanger – 1977
Also termed as chain termination or dideoxy 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 is added instead of the normal
deoxynucleotide, the synthesis stops at that point because the 3’OH is necessary for the
addition of the next nucleotide is absent.
STEPS OF SANGERS METHOD
 NEXT GENERATION TECHNOLOGIES

 Solexa: Based on whole-genome sequencing

 SOLiD (Sequencing by Oligonucleotide Ligation and Detection): Ligation and detection
developed by Life Technologies and has been commercially available since 2006
 454 Pyrosequencing It is a method of high throughput DNA sequencing that utilizes a
single strand of DNA with a length of 400-500 bp.
 FEATURES OF NEXT GENERATION
SEQUENCING
 Highly parallel: many sequencing reactions take place at the same time
 Microscale: reactions are tiny and many can be done at once on a chip
 Fast: because reactions are done in parallel, results are ready much faster
 Low-cost: sequencing a genome is cheaper than with Sanger sequencing
 Shorter length: reads typically range from 505050 -700700700 nucleotides in length
 SHOTGUN SEQUENCING

 is a sequencing method designed for analysis of DNA sequences longer than 1000 base
pairs, up to and including entire chromosomes.
 This method requires the target DNA to be broken into random fragments.
 After sequencing individual fragments using the chain termination method, the sequences
can be reassembled on the basis of their overlapping regions
 USES OF DNA SEQUENCING

Forensics
 Identify individuals
 Determine the paternity of a child
 Identifies endangered and protected species

Medicine
 Detect genes that are hereditary or cause diseases

Agriculture
 Map the genome of microorganism
THE END