DNA Sequencing

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DNA extraction PCR

Gel electrophoresis

Insect identification

ACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGTAGTATTCTTGAATATCAAAAATTTTTGTTGGTTATTCA

DNA sequencingACAGATGTC

TTGTAATCC

GGCCGTTGG

TGGCATAGG

GAAAGGACA

TTTAG

Bioinformatics

Review of Project

How is this organism related to other species?

AF

D

E

B

HI

J

K

??

G

C

2. Amplify and Sequence this region across isolates….

ACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGT

ACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGT

ACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGGTFTGGGTGGATCGATGGCTTATGCTATCGATCAATCAGGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGT

ACAGATGTCTTGTAATCCGGCCGTTGGTGGCATAGGGAAAGGACATTTAGTGAAAGAAATTGATGCGATGGGTGGATCGATGGCTTATGCTATCGATCAATTTAGAATTCAATTTAGAGTACTTAATAGTAGCAAAGGAGCTGCTGTTAGAGCAACACGTGCTCAGGCAGATAAAATATTATATCGTCAAGCAATACGT

Sequence thePCR product

PCR

The elegant idea behind DNA sequencing

Technology changes quickly, but for many years we’ve used Sanger’s cool trick.

Fred Sanger

In the 1970’s, Sanger’s group discovered a fundamentally new method of 'reading' the linear DNA sequence using special bases called chain terminators or dideoxynucleotides. This method is still in use today.

What is the basis of Sanger’s method?

Shared with Walter Gilbert and Paul Berg

DNA Polymerase Action

Mechanism of DNA polymerization

Dideoxy Nucleotide

Dideoxies block elongation

H

ddGTP

X

Sanger Sequencing• Uses DNA polymerase to synthesize a second

DNA strand that is labeled. • DNA polymerase always adds new bases to the

3’ end of a primer that is base-paired to the template DNA.

• chain terminator nucleotides: dideoxy nucleotides (ddNTPs), which lack the -OH group on the 3' carbon of the deoxyribose.

• When DNA polymerase inserts one of these ddNTPs into the growing DNA chain, the chain terminates, as nothing can be added to its 3' end.

Dideoxy (Sanger) Method

• 4 Steps:1. Denaturation

2. Primer attachment and extension of bases (starting point)

3. Termination

4. Gel electrophoresis

Overview: Dideoxy (Sanger) Method

1

4

32

Gel electrophoresis

5

reactions contain the 4 normal dNTPs, but each reaction also contains one of the ddNTPs.

In each reaction, DNA polymerase starts creating the second strand beginning at the primer.

When DNA polymerase reaches a base and the chain will either:

- terminate if a ddNTP is added, or: continue if the corresponding dNTP is added. - which one happens is random, based on ratio of dNTP to ddNTP in the tube

Dideoxy (Sanger) Method

• ddNTP- 2’,3’-dideoxynucleotide• No 3’ hydroxyl• Terminates chain when incorporated• Add enough so each ddNTP is randomly and completely incorporated at each base

Dideoxy Method

• Run four separate reactions each with different ddNTPs• Run on a gel in four separate lanes• Read the gel from the bottom up

DNA Sequencing Reactions

Gel Electrophoretic Fractionationof Products

Automated Version of the Dideoxy Method

Automated sequencers use 4 different fluorescent dyes as tags attached to the dideoxy nucleotides and run all 4 reactions in the same lane of the gel.

Quality of sequence data may vary, depending on:

• Purity and concentration of template DNA • Presence of extra PCR bands (artifacts)• Quality of dye-terminators, electrophoresis matrix, and other reagents

Ideally, look at chromatograms and convince yourself that base calls are robust.

P.S: before coming the class, please watch the link

http://www.youtube.com/watch?v=bEFLBf5WEtc