Matthew 13:17

©2001 Timothy G. Standish

Matthew 13:17

17 For verily I say unto you, That many prophets and righteous men have desired to see those things which ye see, and have not seen them; and to hear those things which ye hear, and have not heard them.


DNA SequencingDNA Sequencing

Timothy G. Standish, Ph. D.


Sequenced GenomesSequenced Genomes Over the past two years large-scale sequencing of

eukaryotic genomes has become a reality Currently the sequencing of at least 4 multicelled

eukaryotic genomes has been completed: 1998 Caenorhabditis elegans - 8 x 107 bp - A

nematode worm 2000 Homo sapiens - 3 x 109 bp - Humans 2000 Arabidopsis thaliana - 1.15 x 108 - A plant

related to mustard 2000 Drosophila melanogaster - 1.65 x 108 bp -

Fruit flies


New TechnologyNew Technology Rapid sequencing of large complex genomes

has been made possible by: Foundational work done over many years

and… Dramatic improvement in DNA sequencing

technology over the past few years In this presentation we will look at both the

basic principles of DNA sequencing and how techniques have been refined to yield the dramatic results we now see


Basic PrinciplesBasic Principles


H

P

O

OH

OH

HO

O

O

CH2

NH2

N

N N

N

Sugar

Base

Phosphate

3’

5’

2’

1’4’

DideoxynucleotidesDideoxynucleotides DNA Sequencing using the Sanger

method involves the use of 2’3’-dideoxynucleotide triphosphates in addition to regular 2’-deoxynucleotide triphosphates

Because 2’3’-dideoxynucleotide triphosphates lack a 3’ hydroxyl group, and DNA polymerization occurs only in the 3’ direction, once 2’3’-dideoxynucleotide triphosphates are incorporated, primer extension stops

H

2’3’-dideoxynucleotide monophosphate

2’-dideoxynucleotide monophosphate

SU

GA

R-P

HO

SP

HA

TE

BA

CK

BO

NE

H

P

O

HO

O

O

CH2

HOH

P

O

O

HO

O

O

CH2

H

P

O

OH

HO

O

O

CH2

NH2

N

N

N

N

O

O

NH2N

NH

N

N

N O

NH2

N

B A

S E

S

2’3’2’3’dideoxy-dideoxy-

nucleotidesnucleotidesTerminateTerminate

DNADNAReplicationReplication OH

P

O

HO

O

O

CH2

HO

O

H 2N

NHN N

N H

H OH

P

O

OH

O

O

CH2

CH 3

O

O

HNN

OH

H

P HO

O

O

CH2

HO

N

O

H 2N

N

H2O

2’3’did

eoxynu

cleotide


DNA SequencingDNA Sequencing In DNA sequencing reactions all the basic components needed to replicate

DNA are used 4 reactions are set up, each containing:

– DNA Polymerase– Primer– Template to be sequenced– dNTPs– A small amount of one ddNTP

ddATP, ddCTP, ddGTP, ddTTP

As incorporation of ddNTPs terminates DNA replication, a series of fragments is produced all terminating with the ddNTP that was added to each reaction


DNA SequencingDNA Sequencing

Plasmid (or phage) with cloned DNA

fragment

Primer Binding sites

Cloned fragment

Primer


The ddATP ReactionThe ddATP Reaction

5’TTATCG3’AATAGCATGGTACTGATCTTACGCTAT5’

5’TTATCGTACCATGACTAGATGCGA

5’TTATCGTACCA

5’TTATCGTACCATGACTA

5’TTATCGTA

5’TTATCGTACCATGA

5’TTATCGTACCATGACTAGATGCGATA

5’TTATCGTACCATGACTAGA

Pol.5’TTATCGTA Let me

Through!

Pol.5’TTATCGTACCATGA

Oh comeon!

Pol.5’TTATCGTACCATGACTAGA

NotAgain!

Pol.5’TTATCGTACCATGACTAGATGCGATA

Agggg….


DNA SequencingDNA Sequencing Products from 4 reactions each

containing a small amount of a dideoxynucleotide are loaded onto a gel

Polyacrlyamide gels capable of separating fragments differing in size by only one base

High concentrations of urea are used to prevent formation of double-stranded DNA or secondary structures

Because polymerization goes 5’ to 3’ shortest fragments are 5’ compared to longer fragments which are in the 3’ direction

ddTTPddCTP ddGTPddATP

Rea

d 5’

to 3

’ fr

om b

otto

m to

top


To read the autorad it is important to start at the bottom and work up so that it is read in the 5’ to 3’ direction

DNA SequencingDNA SequencingWhat A SequencingWhat A Sequencing

Autorad ActuallyAutorad ActuallyLooks LikeLooks Like

A C G T

5’CTAGAGGATCCCCGGGTACCGAGCT...3’


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Matthew 13:17