biotech lect ch04 mod

8/8/2019 Biotech Lect Ch04 Mod

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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 cloning

vector cut with compatible restriction endonucleaseRecombinant 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 ifnecessary


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Cloning Into Bacterial Cells

transformation

Restriction

EndonucleaseDNA Ligase


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DNA Cleavage By Restriction

Endonucleases (1)


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DNA Cleavage By Restriction

Endonucleases (2)


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Recognition Sequences of Restriction

Endonucleases


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Restricti

on

Mappin

g

Mapping Restriction Endonuclease Cleavage Sites


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

digestions of a plasmid


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Restriction Endonuclease

Cleavage Map

Created from single

and multiple enzymedigestions

Useful markers for

noting gene locations

and subcloning

strategies


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Enzymes Used In Recombinant DNA Protocols


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Com

plementar

y

Stic

ky

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 with

selectable markers and

useful cloning sites


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Cloning DNA Into a

Plasmid Vector

Restriction endonuclease

cleave vector/target

Phosphatase vector

Ligate target into vector

Transform into host cells


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Other Plasmid Cloning Vectors

Now too many to count Many 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 codingsequence (e.g. lacZ)


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Antibiotics Commonly Used as Selective

Agents


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Creating and Screening a

Recombinant 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 restriction

endonuclease and cloning the fragmentsinto vectors (plasmid, phage, etc.)

Resultant transformed collection of cells is

called a library


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Partial Restriction Endonuclease

Digestion of DNAs


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Partial Digestion Profile

Collect fragments of a given

target 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 size

of library clones

required to be

screened to find a

single copy gene

varies according to

the genome size ofthe organism to be

studied


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Screening by Hybridization

Probes: DNA or RNA

100+ bp in size good

Sequence match >80% best

Stringency conditions


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Production of Labeled Probes

Random Primer Method


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Three Activities ofE. coli DNAP I (1)

Polymerization of dNTPs

at the 3end of the growing

chain (1)

5exonuclease removes

nucleotides from 5end of

chain immediately

upstream of growing chain

(2) 3exonuclease removes

unpaired nucleotides from

3 end of growing chain

(1)


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Three Activities ofE. 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 tonylon membrane andlysed

DNA binds tomembrane, isdenatured and probehybridized

Bound probe detectedby autoradiographyafter washingmembrane


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Screening by Immunological Assay


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Screening by Functional

Complementation

Requires strain unable

to produce desired

product/function

Cloned DNAs must bein expression vector or

include elements

required for

expression

Select for restoration

of lost function


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Isolation of Poly(adenylated)

mRNAs

Matrix


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cDNA

Synthesis

Oligo(dT) primer

Reverse transcriptase

Klenow/DNAP I RNase H

Degrades RNA ofDNA:RNA hybrid

S1 nuclease Degrades ss nucleic

acids (unpairedloop)


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Enriching for Full

Length cDNAs (1)

Primer has adapter (RE

cutting sequence)

Ribose ends of mRNA arebiotinylated

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 50

kb

Protein coat for efficient

delivery into cells (E.

coli)

Packages DNA 38-52 kb

with cos sites at each end

DNA Replication is by

rolling circle mechanism


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Packaging ofPChromosomes

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

replication

DNA is cleaved at cos and inserted into capsid


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MatureP

Phage DNA packaged in

protein coat

Looks much like alunar lander (actually

has six tail fibers)


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Phage P cloning

vector

Internal segment deleted(now requires helperphage to replicate)

Has cos sites intact Target DNA inserted

between the two Parms (up to about 20kb)

DNA packaged in vitro

Recombinant phageinfectE. coli cells


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Cosmid Cloning System

Pcos sites insertedinto a small

plasmid

Target DNA ligated

between two cosmidDNA molecules

Recombinant DNA

packaged andE. coli

Infected as before

Can clone DNAs up

to 45 kb


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High-Capacity Bacterial Vector

Systems 100-300 kb target size

P1 bacterial systems

F plasmid systems

BACs (bacterial artificial chromosomes


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Genetic Transformation of

Prokaryotes Chemical transformation

Usually involves CaCl2 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 DNA

solution in cuvette

between two electrodes High voltage electric field

pulses administered

DNA migrates through

HVEF induced openings

in 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 >106bp can be transferred in this

manner Can be interspecies

Tripartite mating and multiple selection


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Tripartite Mating

P

. putida difficult totransform

Transform mobilizablerecombinant plasmid

intoE.coli Make culture with P.putida (wt),recombinantE. coli(auxotroph) andE. coli(aux) with conjugativemobilizable plasmid

Recombinant plasmidtransferred to P. putida

biotech lect ch04 mod

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