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Recombinant DNATechnology

Chapter 19

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Recombinant DNA Technology 1971 paper by Kathleen Dana and Daniel

Nathans described isolation of enzyme that

cleaved DNA at specific sequences

1978 Nobel Prize to Nathans, Smith and

Arber for restriction endonuclease

discovery

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Cloning DNA Molecules

Purify DNA from desired source

Cut DNA with restriction endonuclease

Join fragments to cloning vectors cleaved with

compatible restriction endonuclease to createrecombinant DNA molecules

Transfer recombinant molecules to host cells

Isolate DNA from individual clones oftransformed host cells

Do as you please with the isolated DNAsegments

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Restriction Endonucleases

Hundreds now identified

Host cell defense against invading DNAs

Cleave DNA at or near a specific recognitionsequence

Creates restriction fragments

Recognition sites can be from 4 to 8+ base pairs and

are commonly palindromes

First one isolated wasEcoRI fromE. coli

Often produce sticky ends

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Restriction

Endonucleases

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DNA Cleavage ByEcoRI

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Recombinant DNA Molecules

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Cloning Vectors Plasmids

Phage

Cosmids

BACs

YACs

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Plasmids

Circular independent

replicons, origin of

replication (ori)

Generally encode usefulbut not essential genes

E.g. antibiotic resistance or

catabolic pathways

Allow cloning fragmentsup to about 10 kbp

Selectable markers

Multiple cloning sites

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Plasmid Cloning Vectors

As small as possible

with minimum

restriction endonuclease

cutting sites in genes

ori

Selectable marker(s)

Multiple cloning site(MCS)

Reporter function useful

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Cosmids

Plasmid with Pphage

packaging sequence

(cos)

Can clone up to 50 kb Packaged into Pparticles

and injected into host

cells

Circularizes in cell andcontinues as a large

plasmid

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BACs

Bacterial artificial

chromosomes

Can clone up to 200+ kbDNA fragments

Based upon F plasmid

Origin, selectable marker,

promoters to expressed

cloned genes

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YACs

Yeast artificial

chromosomes

Have centromere,telomeres and an

origin of replication,

plus selectable

markers

Cloned segments of

250 kb

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Expression

Vectors Also include

regulatable high level

expression promoter T7 phage promoter

lac operator

lac repressor gene

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First Prokaryotic Host Cells

First clones introduced into strains ofE. coli K12

Protocol

Isolate target DNA

Cut with RE

Ligate to vector

Transform host cells

Plate on antibiotic-containing medium

Identify recombinant plasmids

Identify/characterize specific clones

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Cloning into Plasmids

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Expression of Recombinant

Genes in Eukaryotes

Expression is sometimes desirable in eukaryotic cells

Especially if post-translational modifications are important or

study simply requires it

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Eukaryotic Expression Vectors

Generally similar to prokaryotic ones

Commonly start with prokaryotic vector

Construction done inE. coli

Shuttle vector

Origin

MCS

Selectable marker High expression regulatable promoter

But also generally an intron included

Especially for cDNA clones

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Cloning into Plant Cells

Vectors based upon

Ti plasmid

Derived fromAgrobacterium

tumifaciensplasmid

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Cloning into Mammalian Hosts

Especially for model systems

DNA integrates into a host cell

chromosome

Random vs. site-directed

Transgenics

Models for diseases

Improved individuals???

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Transgenic Mammals

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Polymerase Chain Reaction

PCR

1993Nobel Prize for Kary Mullis

Proposed in 1986 Can provide millions of fold amplification of

a DNA fragment or sequence

Needle in haystack

Revolutionized molecularbiology/genetics/forensics/and everything

Day earth changed

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PCR Procedure

Denature DNA

Add primers, thermostabile

DNAP, dNTPs

Extension

Denaturation

Bind primers

Extension Repeat last 3 steps thirty times

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Chromosome

Sorting Important for early portion

of human genome project

Simplified sequencing effort Fluorescent tags on specific

chromosomes

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Pulse Field Gel Electrophoresis

For very large DNA

molecules

To left are intact yeastchromosomes

Electric field is

pulsed/changed after

very short intervals

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cDNAs Copies of mRNAs in DNA

By reverse transcriptase

Needed to analyze genes and also to express

eukaryotic genes in prokaryotic systems

Introns

cDNA libraries

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cDNA Synthesis

Oligo T primer

Reverse transcriptase and

deoxyribonucleotides

RNaseH

DNAP I makes second

strand using the hairpin

created by reversetranscriptase as a primer

Hairpin cleave by S1

nuclease

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Library Screening

General approach used forboth genomic and cDNAlibraries

DNA from colonies/plaques

transferred to membrane Denature

Hybridize with probe

Detect probe binding

Method depends uponlabeling of probe

Isolate specific probe-binding clones and

culture

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Restriction

Maps

Useful but now

most commonlygenerated by

computer from

actual DNA

sequence

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Southern

Transfer Developed by E. M.Southern

Method for

transferring DNAfrom a gel to a

membrane

Described to right

Also Western and

Northern blots

Proteins and RNA

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Hybridization

Binding of a probe (generally RNA or DNA) to a

nucleic acid in a gel or more commonly bound to amembrane

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DNA

Sequencing

Sanger

Method

describedto right

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Chain Termination DNA

Sequencing

Autoradiograph of results

from Sanger

dideoxyribonucleotide chaintermination method

Sequence is read from bottom

to top

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Fluorescent

Dyes

DNA sequencing with

fluorescent dyes

attached to chain

terminator

dideoxyribonucleotides

Allows for automatedDNA sequencing

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Electropherogram