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Working With Genes2

The document describes the Sanger sequencing method. It involves PCR amplification using a primer and regular dNTPs as well as a small amount of di-deoxy nucleotides, which randomly terminate strand extension. This produces multiple DNA fragments of different lengths that can be separated by electrophoresis to determine the sequence. The termination of strands allows the sequence to be read by determining the length of bases from the primer at each dye-labeled di-deoxy nucleotide position.

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Atharva Purohit
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18 views

Working With Genes2

The document describes the Sanger sequencing method. It involves PCR amplification using a primer and regular dNTPs as well as a small amount of di-deoxy nucleotides, which randomly terminate strand extension. This produces multiple DNA fragments of different lengths that can be separated by electrophoresis to determine the sequence. The termination of strands allows the sequence to be read by determining the length of bases from the primer at each dye-labeled di-deoxy nucleotide position.

Uploaded by

Atharva Purohit
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
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PCR: Template

5’-CGTAATTGTGTACCTGAGTGCCCGTACTGGTAC-3’
3’-GCATTAACACATGGACTCACGGGCATGACCATG-5’
PCR: Melting (94°C)
5’-CGTAATTGTGTACCTGAGTGCCCGTACTGGTAC-3’

3’-GCATTAACACATGGACTCACGGGCATGACCATG-5’
PCR: Annealing (60°C)
5’-CGTAATTGTGTACCTGAGTGCCCGTACTGGTAC-3’

3’-TGACCATG-5’

5’-TGTACCTG-3’
3’-GCATTAACACATGGACTCACGGGCATGACCATG-5’
PCR: Extending (72°C)
5’-CGTAATTGTGTACCTGAGTGCCCGTACTGGTAC-3’
3’-GCATTAACACATGGACTCACGGGCATGACCATG-5’

5’-
TGTACCTGAGTGCCCGTACTGGTAC-3’
3’-GCATTAACACATGGACTCACGGGCATGACCATG-5’
PCR: Melting
5’-CGTAATTGTGTACCTGAGTGCCCGTACTGGTAC-3’

3’-GCATTAACACATGGACTCACGGGCATGACCATG-5’

5’-
TGTACCTGAGTGCCCGTACTGGTAC-3’

3’-GCATTAACACATGGACTCACGGGCATGACCATG-5’
PCR: Annealing
5’-CGTAATTGTGTACCTGAGTGCCCGTACTGGTAC-3’

TGACCATG-5’
5’-TGTACCTG
3’-GCATTAACACATGGACTCACGGGCATGACCATG-5’

5’-
TGTACCTGAGTGCCCGTACTGGTAC-3’

TGACCATG-5’
5’-TGTACCTG
3’-GCATTAACACATGGACTCACGGGCATGACCATG-5’
PCR: Extension
5’-CGTAATTGTGTACCTGAGTGCCCGTACTGGTAC-3’
3’-GCATTAACACATGGACTCACGGGCATGACCATG-5’

5’-
TGTACCTGAGTGCCCGTACTGGTAC-3’
3’-GCATTAACACATGGACTCACGGGCATGACCATG-5’

5’-TGTACCTGAGTGCCCGTACTGGTAC-3’
ACATGGACTCACGGGCATGACCATG-5’

5’-
TGTACCTGAGTGCCCGTACTGGTAC-3’
3’-GCATTAACACATGGACTCACGGGCATGACCATG-5’
PCR: Exponential amplification
• Sequence flanked by primers is amplified
exponentially, the long templates get amplified
linearly
• After 30 cycles, 1 billion copies = 1pg DNA(1000bp)

5’-TGTACCTGAGTGCCCGTACTGGTAC-3’
ACATGGACTCACGGGCATGACCATG-5’
Polymerase Chain Reaction (PCR)

• In-vitro amplification
of specific genes
• Gene fishing for
unknown genes
• Diagnostic primers
for specific variants
• DNA fingerprinting
(AFLP)
Sequencing

Campbell Biology,
Figures 20.13 and 20.12
Reading DNA
• Sequencing:
– Based on copying (PCR).
Reading DNA
• Sequencing:
– Based on copying (PCR).

– dNTP + template + Taq + primer

– Sanger sequencing measures length of product


strand to imply sequence
Sanger sequencing
• Based on a low concentration of di-deoxy
ribonucleotides (with no 3’OH) stopping the
replication process randomly.
Sanger sequencing
• Ribonucleotide

5’

3’
Sanger sequencing
• Deoxy-ribonucleotide

5’

3’
Sanger sequencing
• Di-deoxy ribonucleotide

5’
Sanger sequencing
• Based on a low concentration of di-deoxy
ribonucleotides (with no 3’OH) stopping the
replication process randomly.
5’
3’
Sanger sequencing

5’
3’
Sanger sequencing

5’
3’
Sanger sequencing
• Based on a low concentration of di-deoxy
nucleotides (with no 3’OH) stopping the
replication process randomly.
5’
3’
Sanger sequencing
• Based on a low concentration of di-deoxy
nucleotides (with no 3’OH) stopping the
replication process randomly.
5’
Sanger sequencing
• Based on a low concentration of di-deoxy
nucleotides (with no 3’OH) stopping the
replication process randomly.
5’
Sanger sequencing
• Based on a low concentration of di-deoxy
nucleotides (with no 3’OH) stopping the
replication process randomly.
5’
Sanger sequencing

5’-TGTACCTGAGTGCCCGTACTGGTAC-3’
3’-ACATGGACTCACGGGCATGACCATG-5’
Sanger sequencing: Melting

5’-ACATGGACTCACGGGCATGACCATG-3’

3’-TGTACCTGAGTGCCCGTACTGGTAC-5’
Sanger sequencing: Annealing
only a single primer used

5’-ACATGGACTCACGGGCATGACCATG-3’

3’-GGTAC-5’

3’-TGTACCTGAGTGCCCGTACTGGTAC-5’
Sanger sequencing: Extending
Labelled ddATP stops extension when incorporated

5’-ACATGGACTCACGGGCATGACCATG-3’

ACTGGTAC-5’
Sanger sequencing: Extending
several different sized fragments produced: ddATP

5’-ACATGGACTCACGGGCATGACCATG-3’

ACTGGTAC-5’
AGTGCCCGTACTGGTAC-5’
ACCTGAGTGCCCGTACTGGTAC-5’
Sanger sequencing: Extending
several different sized fragments produced: ddATP

5’-ACATGGACTCACGGGCATGACCATG-3’

ACTGGTAC-5’

AGTGCCCGTACTGGTAC-5’

ACCTGAGTGCCCGTACTGGTAC-5’
Sanger sequencing
A T C G
• Electrophoresis separates
the different length
fragments.
• Only the labeled
fragments are developed.
• Different ddNTPs
resolved in separate
lanes.
Sanger sequencing: Extending
different dNTPs can be labelled with different dyes
A T C G
Sanger sequencing: Extending
different dyes can be run at once

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Sanger sequencing
• Electrophoresis separates the different length
fragments.
• Only the labeled fragments are developed
• Different ddNTPs resolved by different linked
dyes.

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