2. SEQUENCING
• INTRODUCTION
• THE PROCESS OF FIGURING OUT THE CORRECT ORDER OF
THE FOUR NITROGEN-CONTAINING BASES: ⁻
• ADENINE
• GUANINE
• CYTOSINE
• THYMINE
• IN A SECTION OF DNA
• “DETERMINING THE ORDER OF BASES IN A SECTION OF
DNA.”
3. • SANGER SEQUENCING WAS DEVELOPED BY THE
BRITISH BIOCHEMIST FRED SANGER AND HIS
COLLEAGUES IN 1977
• DISCOVERED DNA SEQUENCING BY CHAIN
TERMINATION METHOD
• NOBEL PRIZE 1 (1958)
• COMPLETE AMINO ACID
SEQUENCE OF INSULIN
• NOBEL PRIZE 2 (1980)
• FOR DNA SEQUENCING
SANGER SEQUENCING
4. INGREDIENT
S FOR
SANGER
SEQUENCIN
G
• SANGER SEQUENCING INVOLVES MAKING MANY
COPIES OF A TARGET DNA REGION.
• ITS INGREDIENTS ARE SIMILAR TO THOSE NEEDED
FOR DNA REPLICATION IN AN ORGANISM, OR FOR
POLYMERASE CHAIN REACTION (PCR), WHICH
COPIES DNA IN VITRO.
• THEY INCLUDE:
• A DNA POLYMERASE ENZYME
• A PRIMER, WHICH IS A SHORT PIECE OF SINGLE-
STRANDED DNA THAT BINDS TO THE TEMPLATE
DNA AND ACTS AS A "STARTER" FOR THE
POLYMERASE
5. INGREDIENTS FOR SANGER
SEQUENCING
• THE FOUR DNA NUCLEOTIDES (DATP, DTTP,
DCTP, DGTP)
• THE TEMPLATE DNA TO BE SEQUENCED
• HOWEVER, A SANGER SEQUENCING REACTION
ALSO CONTAINS A UNIQUE INGREDIENT:
• DIDEOXY, OR CHAIN-TERMINATING, VERSIONS OF
ALL FOUR NUCLEOTIDES (DDATP, DDTTP, DDCTP,
DDGTP), EACH LABELLED WITH A DIFFERENT
COLOR OF DYE
6. DIDEOXY NUCLEOTIDES
• LACK AN -OH GROUP AT THE 3-CARBON
POSITION
• CANNOT ADD ANOTHER NUCLEOSIDE AT
THAT POSITION
• PREVENT FURTHER DNA SYNTHESIS
7. DIDEOXY
NUCLEOTID
ES
• INCORPORATION OF A DIDEOXYNUCLEOTIDE TO
GROWING DNA STRAND TERMINATES ITS FURTHER
EXTENSION
• ARE ADDED IN SMALL PROPORTION
• DATP DDATP
• DGTP DDGTP
• DCTP DDCTP
• DTTP DDTTP
8. METHOD
OF DNA
SEQUENCIN
G
• TO SEQUENCE THE DNA:
• SEPARATE THE DNA INTO TWO STRANDS.
• THE STRAND TO BE SEQUENCED IS COPIED USING
CHEMICALLY ALTERED BASES.
• THESE ALTERED BASES CAUSE THE COPYING
PROCESS TO STOP
• EACH TIME ONE PARTICULAR LETTER IS
INCORPORATED INTO THE GROWING DNA CHAIN.
• THIS PROCESS IS CARRIED OUT FOR ALL FOUR
BASES.
9.
10. APPLICATIO
NS OF
SANGER
SEQUENCIN
G
• SANGER SEQUENCING GIVES HIGH-QUALITY
SEQUENCE FOR RELATIVELY LONG STRETCHES OF
DNA UP TO 900 BASE PAIRS
• IT'S TYPICALLY USED TO SEQUENCE INDIVIDUAL
PIECES OF DNA
• SUCH AS BACTERIAL PLASMIDS OR DNA COPIED
IN PCR.
11. APPLICATIO
NS OF
SANGER
SEQUENCIN
G
• APPLICATIONS OF DNA SEQUENCING
INCLUDE SINGLE NUCLEOTIDE
POLYMORPHISM (SNP) DETECTION.
• SINGLE-STRAND CONFORMATION
POLYMORPHISM (SSCP)
• HETERODUPLEX ANALYSIS
• AND SHORT TANDEM REPEAT (STR) ANALYSIS
13. LIMITATION
S OF
SANGER
SEQUENCIN
G
• SANGER SEQUENCING HAS A NUMBER OF
LIMITATIONS THAT CAN LEAD TO PROBLEMS WITH
RESULTS AND DIFFICULTY USING THE METHOD IN
GENERAL:
• SANGER METHODS CAN ONLY SEQUENCE SHORT
PIECES OF DNA--ABOUT 300 TO 1000 BASE
PAIRS.
• THE QUALITY OF A SANGER SEQUENCE IS OFTEN
NOT VERY GOOD IN THE FIRST 15 TO 40 BASES
BECAUSE THAT IS WHERE THE PRIMER BINDS.
• SEQUENCE QUALITY DEGRADES AFTER 700 TO
900 BASES.
14. LIMITATION
S OF
SANGER
SEQUENCIN
G
• IF THE DNA FRAGMENT BEING SEQUENCED HAS
BEEN CLONED, SOME OF THE CLONING VECTOR
SEQUENCE MAY FIND ITS WAY INTO THE FINAL
SEQUENCE.
• COST EFFECTIVENESS
• SANGER SEQUENCING IS RELATIVELY EXPENSIVE,
RUNNING ABOUT $500 PER 1000 BASES,
COMPARED TO LESS THAN $0.50 PER 1000 BASES
FOR NEXT GENERATION SEQUENCING,
ACCORDING TO A 2011 STUDY.
15. LIMITATION
S OF
SANGER
SEQUENCIN
G
• SANGER MAY NOT BE ACCURATE FOR VALIDATION
• SANGER SEQUENCING IS WIDELY USED TO
VALIDATE NEXT GENERATION SEQUENCING
RESULTS BECAUSE SANGER SEQUENCING IS
CONSIDERED TO BE MORE ACCURATE. HOWEVER,
A RECENT STUDY FROM THE NATIONAL HUMAN
GENOME RESEARCH INSTITUTE SHOWS THAT MAY
NOT BE TRUE. THE GROUP OF RESEARCHERS
COMPARED DNA SAMPLES FROM A GENOMIC
SEQUENCING PROJECT THAT HAD BEEN ANALYSED
BY BOTH NEXT GENERATION AND SANGER
METHODS.
