the techniques of molecular genetics: recombinant...

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The techniques of moleculargenetics: Recombinant DNA

I. Introduction to Recombinant DNAtechnology

II. Restriction EnzymesIII. VectorsIV. Molecular Techniques

I. Introduction to RecombinantTechnology

A. What is a recombinant? Recombinant DNA refers to a combination of DNA molecules

that are not found together in nature, produced by joining DNAobtained from different biological sources

• Vector =

Permit entry into host cell, where it can be replicated or clonedinto many copies

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Recombinant DNA technologyB. Making

recombinants =

1) DNA is isolated & purified2) Restriction Enzymes

generate fragments3) Fragments inserted into

vector4) Vector transferred to host

cell5) As host cell replicates,

recombinant moleculesare passed on to progeny

6) Cloned DNA can berecovered & analyzed –can be transcribed &translated in the host cell

II. Restriction Enzymes• restriction enzyme binds to DNA at a specific recognition

sequence and

Host cell protects its DNA via methylation

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EcoRI

PstI

SmaI

RE’srecognizespecificPalindromicsequences=recognitionsequences

cleavageinoffsetmannerproducesoverhangsthatprovide

Properties of REs• Named after the bacteria from which they were isolated

– EcoRI = from E. coli, it’s in group I

• Three groups based upon the types of sequences theyrecognize, the nature of the cleavage made in the DNA

• Blunt & Staggered Cuts SmaI = blunt cut EcoRI = staggered cut (leaves a 5’ overhang) PstI = staggered cut (leaves a 3’ overhang)

• DNA ligation = in staggered cuts, if two compatible endsanneal, then DNA ligase seals the phosphodiester bondbetween the two DNA molecules

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1).CreateDonorDNAfragment,viarestrictiondigest

2).Addfragmenttoavector,stickyendshybridizetovectorandarepastedusingligase

3).DonorDNAisreplicated,transcribedandtranslated,andthustherecombinantscanbeselected.

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makingclones

III. Types of Vectors Cloning vectors Must have:

A. Plasmids (5-10 kb)B. Bacteriophage (10-40 kb)C. Cosmids (50 kb)D. BACs & YACs (300 kb, up

to 1,000 kb)

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A. Plasmids used for DNA cloning usually have been engineered to contain: • a number of convenient restriction sites • a selectable marker gene to select for its presence in the host cell

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B. phage vector• The central third of lambda (λ) phage

vectors can be replaced with foreignDNA without affecting the ability toinfect cells and replicate 1000X more efficient than plasmids

• Cosmid vectors are created by combining partsof λ phage and parts of plasmids.

• Cosmids contain the cos sites of lambda, whichare necessary for packaging of phage DNA intophage particles.

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D. Artificial chromosomes - BACs & YACs• Bacterial artificial chromosomes (BACs) are based on F factor and can carry

up to 300 kb of inserted DNA• Yeast artificial chromosomes (YACs) can contain

IV. Construction & screeninggenomic libraries

A. Overview of genomic libraries:DNA library

– Pool of all recombinant plasmids generated by ligatingDNA fragments from a source of interest into a vector

cDNA library– contains complementary DNA copies made from the

mRNAs present in a cell population and represents thegenes that are

– Isolated mRNA– This is then cloned into a vector to make smaller a cDNA

library.

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B. Finding specific DNA moleculesfrom a library (screening)

1) Selecting for cloning vectors– You can plate the bacteria with a particular gene on a

selective plate

2)Probingforclonesofaspecificgene:ColonyHybridization

Labeledprobe

Autoradiographyusedtodetecthybridization.

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Onceaclonedsequencehasbeenidentifiedandselectedfromalibraryitcanbeusedformanythings:

probetofind/studyregulatoryregionsinvestigateorganizationofthegenestudyexpressionincells/tissues

AnalyzingClonedSequences:

V. Molecular Techniques

A. Restriction Digests (RFLPs)B. PCRC. DNA Sequencing

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A. Restriction Fragment LengthPolymorphisms (RFLPs)

Variations in DNA fragment length generated bycutting with a restriction enzyme

Restriction mapping = A restriction map establishesthe number and order of restriction sites and thedistance between restriction sites on a cloned DNAsegment

Inherited as alleles & can be mapped to specificregions on individual chromosomes (used asmarkers)

200 bp 50bp 400bp 100 bp

Restriction MappingRestriction Mappinglook at the number orderlook at the number orderand distance betweenand distance betweenenzyme cutting sitesenzyme cutting sitesalong a cloned segmentalong a cloned segmentof DNAof DNA

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B. The Polymerase Chain Reaction Makes DNA Copies without Host Cells

PCR requires two oligonucleotide primers, one complementary to the 3'end of one strand of the DNA to be amplified and one complementary to the3' end of the other strand.primers anneal to denatured DNA, and the complementary strands aresynthesized by a heat-stable DNA polymerase

C. DNA sequencing

process for sequencingparticular regions of DNA, 4sequencing reactions made foreach base

sequencing is automated anduses fluorescent dye-labeleddideoxynucleotides

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Chain termination method1) primer annealed to a singlestrand of DNA (3’end) this isdistributed into 4 tubes. Eachtube has one of the 4 dNTPsmodified as dideoxynucleotide(ddNTP) w/ label.2) DNA pol is added to eachtube & the primer is elongatedforming a complementary strandto the template.3) As synthesis takes place, thepol can insert a ddNTP insteadof a dNTP, causing synthesis toSTOP. As the reactionproceeds, the tube w/ ddATPwill accumulate molecules thatterminate at all positionscontaining A.4) The DNA fragments fromeach reaction tube areseparated by gel electrophoresis& read from bottom to top!