Dna sequencing scenario
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DNA Sequencing Scenario. DNA replication and chain termination. ddTTP No OH group = chain termination at T residue. dATP. O H. Ö H. H. 5 ´. T. P. P. P. P. P. P. P. P. P. P. C. T. A. G. A. A. G. T. C. T. A. T. C. P. P. P. P. P. P. P. P. 3 ´.

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DNA Sequencing Scenario

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Dna sequencing scenario

DNA Sequencing Scenario


Dna sequencing scenario

DNA replication and chain termination

ddTTP

No OH group = chain termination at T residue

dATP

OH

ÖH

H

T

P

P

P

P

P

P

P

P

P

P

C

T

A

G

A

A

G

T

C

T

A

T

C

P

P

P

P

P

P

P

P

Adapted from Lehninger Principles of Biochemistry 4th Edition

Primer strand

The 3´ hydroxyl (OH) group interacts with the phosphate group on the incoming nuclueotide to form a phosphodiester bond

Template strand


Dna sequencing scenario

Nucleosides & Nucleotides

Taken from: http://en.wikipedia.org/wiki/Nucleotide

Nucleoside = a heterocyclic base (A, G, C, U or T) and a five carbon sugar (pentose) either ribose for RNA or deoxyribose for DNA

Nucleotide = a heterocyclic base and a pentose sugar with one or more phosphate groups (mono-, di- or tri-phosphate).

Nucleotides are the monomers of nucleic acids, with three or more bonding together in order to form a nucleic acid.


Dna sequencing scenario

Base

O

H

H

H

H

O-

O-

O-

O-

O-

O-

O-

O-

O-

OH

OH

P

P

P

-O

O

P

O

P

O

CH2

-O

-O

O

O

P

P

O

O

P

P

O

O

CH2

CH2

O

O

O

O

O

O

O

O

O

Base

O

H

H

H

H

OH

H

Base

O

H

H

H

H

H

H

Nucleotide structure continued

Nucleoside tri-phosphate (NTP)

With a ribose sugar (OH at C2 & C3) used to synthesise RNA.

Deoxynucleoside tri-phosphate (dNTP)

With a deoxyribose sugar(OH at C3) used to synthesise DNA.

Dideoxynucleoside tri-phosphate (ddNTP)

With a dideoxyribose sugar(no OH groups) used for the Sanger method (dideoxy) sequencing.


Dna sequencing scenario

phosphodiester bond

http://en.wikipedia.org/wiki/Phosphodiester_bond

Nucleotides are joined together by phosphodiester bonds

Phosphodiester bonds require a hydroxyl group (OH) to be present on the 3´ carbon to react with a phosphate on in 5´ carbon of the sugar residue of the next nucleotide.

If no 3´ hydroxyl group is available no further nucleotides can join the DNA strand and the chain terminates.This is how dideoxynucleotides prevent chain elongation – they lack a 3´ OH group on the sugar.


Dna sequencing scenario

  • Components of the reaction mix

  • DNA template

  • DNA primer

  • DNA polymerase -Taq polymerase

  • Mixture of deoxy (dNTPs) & fluorescently labelled dideoxy nucleotides (ddNTPs)

Computer analysis – base calling

Outline of Fluorescent Dideoxy DNA Sequencing

20 – 35 cycles

Apply to a capillary gel & subject to electrophoresis

The fragments are detected after excitation of the labelled ddNTPs by a laser

Fluorescent peaks and bases are visualised and analysed


Dna sequencing scenario

T

TC

PRIMER

TCAGAT

PRIMER

PRIMER

PRIMER

PRIMER

PRIMER

PRIMER

PRIMER

TCA

TCAG

TCAGATAG

TCAGA

TCAGATA

The sequencing reaction continued

Components in the reaction mix

Taq polymerase

OH

PRIMER

+ dATP, dCTP, dGTP, dTTP,

+ ddATP, ddCTP, ddGTP, ddTTP

AGTCTATATC

TEMPLATE

Termination products obtained

Remember that the sequence obtained with be complementary to the template sequence!

Therefore our new 5´  3´ complementary sequence to our original template sequence reads TCAGATAG.


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