21.recombinant dna technology[1]

8/14/2019 21.Recombinant DNA Technology[1]

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Recombinant DNA Technology ILodish Chapters 5.2

it all began with the discovery of the bacterial defense system

that RESTRICTS phage growth. In the late 1960s, Stewart Linn

and Werner Arber discovered two classes of enzymes: methylases

and RESTRICTION endonucleases.

At the same time, Charles Richardson had purified DNA ligase of

the E.coli phage T4.all you needed to do was to cut and ligate..

And thats what Paul Berg did in the 70sand he received the

Nobel Prize in 1980.


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Restriction enzymes cut DNA molecules

at specific sequences


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Most restriction enzymes recognize short palindromes and

cut unmethylated DNA

Frequency of 6 cutters: 46 = once every 4096 bp

Frequency of 4 cutters: 44 = once every 256 bp


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Today we know more than 600 different

restriction endonucleases


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DNA cloning with plasmid vectors

Recombinant DNA technology depends on the abilityto produce large numbers of identical DNA molecules(clones)

Clones are typically generated by placing a DNAfragment of interest into a vector DNA molecule,which can replicate in a host cell

When a single vector containing a single DNA

fragment is introduced into a host cell, large numbersof the fragment are reproduced along with the vector


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Plasmid vectors have an ori, a resistance marker

and a multi-cloning site (polylinker)


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and 2) transform E.coli to multiply DNA


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Identical E.coli clones carry identical (cloned) DNA

Usually great to clone short fragment of a few kb


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Complementary DNA (cDNA) libraries

are prepared from isolated mRNAs


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Preparation of a cDNA library


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How do you identify a clone that

carries a specific gene?


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How will I obtain the sequence of my cDNA?

The Sanger (dideoxy) method


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Polymerase chain reaction: an alternative

to cloning

The polymerase chain reaction (PCR) can be used toamplify rare specific DNA sequences from a complexmixture when the ends of the sequence are known

PCR comes in two flavors: 1) DNA template based,or 2) RNA template based (reverse transcriptasePCR)

.Kary Mullis, surfing father of PCR sold the technologyto Cetus for $10,000. Cetus sold the technology for a

stunning $300,000,000 a few years later

Mullis received the Nobel Prize in 1993 and turned his back

on both academia and industry.


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Polymerase chain reaction

thermostable

polymerase


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PCR products can be cloned into vectors (e.g., for protein expression)

R i PCR


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RNase H

gene-specific primer

add second gene-specific primer

to amplify (by regular PCR)

Reverse transcriptase PCR(can be used in clinic to probe patient sample for oral pathogens)

Start with single strandedtemplate (mRNA)


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Transposon mutagenesis

This strategy is widely used

to create mutants in bacteria

and allows for easyidentification of the

disrupted gene.


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21.recombinant dna technology[1]

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