Biotech Lect Ch05 Mod

8/9/2019 Biotech Lect Ch05 Mod

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Chemical

Synthesis of DNA (1)

Automated multistep

process Commonly

phosphoramidite

chemistry



Automated DNA Synthesis (2)

First nucleotide

attached to matrix

DMT is a blockinggroup which is

removed before the

first coupling cycle



Automated DNA Synthesis (3)

Phosphoramidite

nucleotide

3¶phosphite end isthe reactive portion

DMT blocks 5¶end



DNA Synthesis (4): Coupling Reaction



DNA Synthesis (5): Capping

³Permanently

blocks unreacted

sites Prevents N-1

length products



DNA Synthesis (6) Oxidation

Phosphite triester

linkage is oxidized

to pentavalent phosphate triester

Entire cycle can

now be repeated



Overall Yield of Chemically Synthesized

Oligonucleotides for Various CouplingEfficiencies for Each Cycle



Nucleotide Sequences Deduced

from Amino Acid Sequences

Example A shows one possible

nucleotide sequence for a three

amino acid sequence, whileFigure B shows the effect of

degenerate sites on the possible

number of oligonucleotide

sequences to be made



Linkers and Adapters

Linkers are generally

double stranded and

blunt ended

oligonucleotidescontaining a restriction

enzyme cutting site

Adapters generally

change a specific endfrom restriction enzyme

cleavage to another

enzymes cleavage site



Cloning With Linkers

Fragments from

blunt cutting RE



Adding RE Sites Using an Adaptor

New site in vector

Sticky ends on fragment



Assembly of a Synthetic Gene (1)

Synthesize

overlapping

polynucleotideswhich collectively

encompass entire

gene

Ligate using T4DNA ligase



Assembly of a

Synthetic Gene (1)

Synthesize overlapping

polynucleotides which

leave ss gaps in gene

Fill gaps using DNAP I

Ligate with T4 DNA

ligase



DNA Sequence Determination (1)

Fred Sanger ± chaintermination method

A normal

(2¶deoxyribonucleotide)

and a 2¶,3¶-dideoxyribonucleotide

(chain terminator)




Normal DNA

synthesize using

2¶deoxyribonucleotide Chain elongation still

possible on 3¶end




Incorporation of a

2¶,3¶dideoxyribonucleotide Further chain elongation

not possible due to lack of

free 3¶OH group



Primer Extension

DuringDideoxyribonucleotide

DNA Sequencing

Reactions

2. Note that all fragments of a

given length end with thesame dideoxyribonucleotide

1. Primer sets start point for all

fragments synthesized

3. With color-coded dyes all

four reactions are possible in

one tube



DNA Sequence Determination (5):

Electrophoretic Sizing Fragments are sized

by electrophoretic

migration Gel electrophoresis

Capillary gel

electrophoresis

Actual sequenceshown at right



Automated DNA Sequencing

Laser dyes

Capillary gel

electrophoresis Fluorescent detection

Example of ³old´

autoradiogram with

radioactive productsshown at bottom



M13 Cloning and Sequencing Vector

ssDNA phage with dsDNA

replication form

Cloning into ds form

Phage modified to have MCSand lacZ reporter gene

Cloning into MCS inactivates

B-galactosidase gene (no X-

gal to blue conversion

Commonly use ss forms for

DNA sequencing templates



Cloning Into M13

Vectors (2) Available in

M13mp18 and M13

mp19 forms MCS reversed

Allows ss templates

for sequencing both

strands of targetduplex to be

produced



Primer Walking

Synthesize secondsequencing primer from3¶end of new sequencedetermined

Repeat as necessary toobtain full lengthsequence

Each step ³normally´

advances abut 500 or so base pairs

Complementary strandshould also be completed



Polymerase

Chain Reaction

(PCR) Allows 108+fold

amplification of specific

DNA sequences Procedure

± Denature DNA

± Anneal primers flanking

region to be amplified

± Extend primers using

T aq DNA polymerase I

± Repeat cycle 30-40 times



Polymerase

Chain Reaction After 1 cycle copies

double

After 2 cycles copieddoubled and new

strand is unit length

(goes only from

primer to primer)



Polymerase

Chain Reaction

After 3 cycles copies are

increased 8-fold and new

copies are ds and unitlength

Odd length copies

increase arithmetically

Unit length copies

increase in number

geometrically



Polymerase Chain Reaction By completion of

30-40 cycles nearly all

copies are ds DNAand unit length

products



Gene Synthesis by PCR (1)

Used to amplify genes too largefor single step amplification

With more processive enzymes

more commonly used today to fuse

5¶ and 3¶ ends of related butdifferent genes or to carry out other

types of genetic engineering



Gene Synthesis by PCR (2)



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Biotech Lect Ch05 Mod