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DNA Sequencing
Two Techniques Approached GROUP 1 Ismail bin Rajuli 155954 Mohd Aidil Hanan bin Jusoh 156028 Vinodhini Krishna Rao 160406 Sri Nur Aimi bte Azmi 159118 Nur Atifah bteedit Master subtitle157002 Click to Aziz style Fatin Hanani

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Lets Get An Idea

DNA SEQUENCING

Biochemical methods used to map out the sequence of the DNA nucleotides of the DNA into a format that is decipherable by the order of the nucleotide bases:Adenine Guanine Cytosine Thymine

Translation

Determined

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The Ideas
DNA

sequencing invention began with discoveries of double-helix structure in early 1950s first sequencing was invented in the late 1960s modern sequencing techniques were invented in the late 1970s

The

Two

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History on The Common Two

Sanger

and Coulson plus-minus sequencing

> only for ssDNA

Maxam

and Gilbert chemical sequencing

> requires series of complex reaction

Sanger

et al chain-termination

> idea of inhibitor

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Sangers dideoxy principle (1977)

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Techniq ue

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1.

DNA segment of interest is heated to disassociated the strands. To seq. DNA fragment, Each of 4 Rxn Tubes prepared with

1.

. . . .

ssDNA template DNA polymerase 32P labelled primer Small amount of different ddNTP (ddCTP,ddGTP,ddATP,ddTTP) 4 normal dNTP

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ddNTP

dNTP

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3.

A ddNTP will be incorporated randomly, at different sites in different synthesis in the reaction tube. In each rxn, different chain length produced at respective ddNTP that terminated chain growth.

3.

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5.

Sample in 4 rxn tube loaded onto adjacent lanes on a sequencing gel. Run gel electrophoresis, then exposed to film to produce autoradiogram. Base seq. determined by scanning up the gel. DNA
migration

Lane

5.

5.

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Automated Sangers principle (Sanger derivative method, 1990)

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Techniq ue

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1.

Used robotic & computer system. DNA SEQ. carried out in single rxn: Template DNA All 4 normal dNTP (deoxy) dA dd All 4 flourescently labelled ddNTP TP dd GT (dideoxy) ATP dG P ddC (ddCTP,ddGTP,ddATP,ddTTP) TP TP ddT dCT Unlabelled primer & DNA P dT TP polymerase I with buffer TP

1. . . .

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3.

Incubate the reaction for a period. The fragments are separeted by length from longest to shortest using polyacrylamide gel

3.

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5.

A laser constantly scans the bottom of the gel, detecting bands as they move down the gel. Each of the 4 ddNTP flouresces at different color when illuminated by a laser beam.

5.

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An

automatic scanner provides a printout of the sequence.

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Advantages
DNA sequences Sanger method Maxam-Gilbert Simple no

are method preliminary obtained from the extension is original DNA required, and not molecule therefore avoiding incubation from a synthesized and copypurification. commercially sequencing of small availableDNAPoly fragments. meraseI.

Permits direct It requires

The

results are better with fewer artifacts and a larger

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Disadvantages
Technical complexity Sanger method Maxam-Gilbert There are occasional

method artifacts, likely due prohibiting its use in to contaminant standard molecular fragments biology kits, extensive use of Band pile-ups can hazardous occur due to loop chemicals, and formation under gel difficulties with conditions, and is scale-up. usually depicted as Chemicals bands in numerous used are the same position, or toxic very close together Methods are labor on the gel. intensive

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CONCLUSION
Maxam-Gilbert

sequencing technique gives different bands of nucleotide according to the mixture of purines and pyrimidines. sequecing technique gives specific bands of nucleotide sequence. what technique you want to use.

Sanger

Depend