2.Biotech Lect

8/14/2019 2.Biotech Lect

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Recombinant DNA Technology

Common General Cloning Strategy Target DNA from donor organism extracted, cut with

restriction endonuclease and ligated into a cloningvector cut with compatible restriction endonuclease

Recombinant construct transferred into host cell Host cells which do not take up construct are

eliminated by selection protocol

Host cell library screened to identify desired clone if necessary


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Cloning Into Bacterial Cells

transformation

RestrictionEndonuclease

DNA Ligase


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DNA Cleavage By Restriction

Endonucleases (1)


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DNA Cleavage By RestrictionEndonucleases (2)


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Recognition Sequences of RestrictionEndonucleases


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Restriction

Mapping

Mapping Restriction Endonuclease Cleavage Sites


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DNA fragment sizes (in kilobase pairs) after single and double restriction endonucleases

digestions of a plasmid


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Restriction EndonucleaseCleavage Map

Created from single

and multiple enzymedigestions

Useful markers for noting gene locationsand subcloningstrategies


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Enzymes Used In Recombinant DNA Protocols


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Com

plementar

ySticky

Ends

Annealing of Complementary Sticky Ends


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T4 DNA Ligase Action


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Early Cloning Vectors

pBR322 Plasmid

Small independentreplicon withselectable markers anduseful cloning sites


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Cloning DNA Into aPlasmid Vector

Restriction endonucleasecleave vector/target

Phosphatase vector Ligate target into vector Transform into host cells


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Other Plasmid Cloning Vectors

Now too many to countMany specialized for expression, etc.

pUC series Multiple cloning sites Improved reporter/selection genes


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Multiple Cloning Sites

Synthetic oligonucleotide construction Polymer of cutting sites Can be included in reporter gene coding

sequence (e.g. lacZ)


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Antibiotics Commonly Used as SelectiveAgents


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Creating and Screening aRecombinant DNA Library

A library is a collection of subdivided portions of a larger genetic element or

genome Commonly created by partial digestion of genomic DNA with restrictionendonuclease and cloning the fragmentsinto vectors (plasmid, phage, etc.)

Resultant transformed collection of cells iscalled a library


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Partial Restriction EndonucleaseDigestion of DNAs


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Partial Digestion Profile

Collect fragments of a giventarget size after digestions for different times or using

different restrictionendonuclease concentrations Size fractionate and combine

fractions of desired target size


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Genome Sizes of Various Organisms

The number and sizeof library clonesrequired to bescreened to find asingle copy genevaries according to

the genome size of the organism to bestudied


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Screening by Hybridization

Probes: DNA or RNA100+ bp in size goodSequence match >80% best

Stringency conditions


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Production of Labeled Probes

Random Primer Method


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Three Activities of E. coli DNAP I (1)

Polymerization of dNTPsat the 3end of the growingchain (1)

5exonuclease removesnucleotides from 5end of chain immediatelyupstream of growing chain(2)

3exonuclease removesunpaired nucleotides from3 end of growing chain

(1)


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Three Activities of E. coli DNAP I (2)

(2)

(3)

Note that the 5exonuclease is used in nick translation and the 3exonuclease activity is used for the proofreading function


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Screening Colonies by Hybridization

Nucleic acid probe Cells transferred to

nylon membrane andlysed

DNA binds tomembrane, isdenatured and probehybridized

Bound probe detected by autoradiographyafter washingmembrane


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Screening by Immunological Assay

b l


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Screening by FunctionalComplementation

Requires strain unableto produce desired

product/function

Cloned DNAs must bein expression vector or include elementsrequired for expression

Select for restorationof lost function


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Isolation of Poly(adenylated)mRNAs

Matrix


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cDNASynthesis

Oligo(dT) primer Reverse transcriptase Klenow/DNAP I RNase H

Degrades RNA of DNA:RNA hybrid

S1 nucleaseDegrades ss nucleicacids (unpairedloop)


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Enriching for FullLength cDNAs (1)

Primer has adapter (REcutting sequence)

Ribose ends of mRNA are biotinylated RNase I degrades ss RNA Only full length cDNA is

still attached to a biotinylated mRNA(biotin still on 5end)

Capture full length copies

adapter


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Enriching for Full Length cDNAs (2)

RNase H degrades mRNA Add poly(G) to cDNA

Primer/Adapter with oligo(C) DNAP I (Klenow) Restriction endonucleases Cut Vector DNA Ligase Transform


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Inert Capacities Common Vector

Systems


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Bacteriophage Lambda Life Cycle Lysogenic phage

Lysogeny vs. lytic cycle

Chromosome about 50kb

Protein coat for efficientdelivery into cells ( E.coli )

Packages DNA 38-52 kbwith cos sites at each end

DNA Replication is byrolling circle mechanism


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Packaging of l Chromosomes

Natural DNA is concatemer with cos sitesseparated by about 50 kb (from rolling circlereplication

DNA is cleaved at cos and inserted into capsid


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Mature l Phage

DNA packaged in protein coat

Looks much like alunar lander (actuallyhas six tail fibers )


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Phage l cloningvector

Internal segment deleted(now requires helper

phage to replicate) Has cos sites intact Target DNA inserted

between the two l arms (up to about 20kb)

DNA packaged in vitro Recombinant phage

infect E. coli cells

Cosmid Cloning System


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Cosmid Cloning System

l cos sites insertedinto a small

plasmid

Target DNA ligated

between two cosmidDNA molecules

Recombinant DNA packaged and E. coliInfected as before

Can clone DNAs upto 45 kb


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High-Capacity Bacterial Vector Systems

100-300 kb target sizeP1 bacterial systems

F plasmid systemsBACs (bacterial artificial chromosomes


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Genetic Transformation of Prokaryotes

Chemical transformationUsually involves CaCl 2 and heat shock

Transformation frequency about 1/1000 Electroporation

Electric field meidated membrane

permeabilization 10-100 times more efficient that chemical

approach

Much better for large plasmids (100+ kb)


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Electroporation

Cells suspended in DNAsolution in cuvette

between two electrodes High voltage electric field pulses administered

DNA migrates throughHVEF induced openingsin cells


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Conjugation

Natural system of transmitting plasmids fromone cell/strain to another

Requires specific DNA sequences on

transferred plasmid and certain proteins whichcan be provided in trans Plasmids of >10 6 bp can be transferred in this

manner Can be interspecies Tripartite mating and multiple selection


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Tripartite Mating P. putida difficult to

transform Transform mobilizable

recombinant plasmidinto E.coli

Make culture with P. putida (wt),recombinant E. coli (auxotroph) and E. coli (aux) with conjugativemobilizable plasmid

Recombinant plasmidtransferred to P. putida


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Thanking you

Er. Ashok Kumar 9450501471,9816170568

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2.Biotech Lect