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Block II Lecture 1: Recombinant DNA Technology

Part I. DNA Manipulations: Basic Techniques

Overview of the ProcedureCloning VectorsTarget Gene Selection and Acquisition

Restriction EndonucleasesPolymerase Chain Reaction (PCR)

DNA Ligation, Transformation, and SelectionClone Identification and Screening

Restriction Digestion Analysis

Thermal Cycle DNA SequencingLibrary Construction and AnalysisShotgun Approaches for Sequencing Genomic DNA


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Cloning: To make identical copies

DNA cloning involves separating a specific gene or DNA segmentfrom a chromosome, attaching it to a DNA carrier molecule, andreplicating this modified DNA, thousands or millions of times,through an increase in cell number and DNA copies per cell.

The result is selective purification and amplification of a particulartarget gene or DNA segment from a complex mixture of DNAmolecules.

The methods used to accomplish these and related tasks arecollectively referred to as recombinant DNA technology orgenetic engineering.


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Developed fromnaturally occurringbacterial plasmids

Contain an origin ofreplication (ori)

Contain numerous

restriction sites

Contain genes thatconfer resistance toantibiotics, thusallowing selection ofbacterial coloniescarrying the plasmid

Introduced into

competent bacterialcells by transformation

Cloning vectors allow amplification of inserted DNA fragments


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Different types of cloning vectors

Plasmids: Circular DNA molecules which replicate separatelyfrom the host chromosome. Plasmids used for genomic andcDNA cloning. Bacterial host. Insert size range < 15kb.

Bacteriophage-based Cosmids:Linear DNA moleculesused for genomic and cDNA cloning. Bacterial host. Insert sizerange < 20kb.

Bacterial Artificial Chromosomes (BACs): Circular DNA moleculesused for cloning very long segments of genomic DNA. Bacterialhost. Insert size range 100-300 kb.

Yeast Artificial Chromosomes (YACs) : Specialized DNA moleculesused for cloning very, very long segments of genomic DNA.Yeast host. Insert size range 100-2000kb.


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Mammalian expression vector

Note : MultipleCloning Sites (MCS)or a Polylinker Region

With theexceptionof buddingyeast,plasmids areuncommonin eukaryotes.Thus, mosteukaryoticvectors arebased on DNAor RNA viralgenomes.

*

*

**

*

* * Viral DNA sequences

Bacterial sequences


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A restriction enzymes binds to DNA at a specific sequence and make a double-strandedcut at or near that sequence.

Restriction endonucleases cut DNA molecules at defined positions


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Blunt and sticky ends

5 and 3 overhangs

The same sticky ends produced by different enzymes

Digestion of DNAwith different

restrictionendonucleases


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Polymerase Chain Reaction (PCR)

DNA from a selected region of the chromosome or genome can to be amplified a billion-fold,effectively purifying it away from a complex mixture of DNA molecules.

REQUIREMENTS:

Oligonucleotide primers which

flank the sequence of interest

A DNA Template (a few ng)

A thermal-stableDNA Polymerase (TAQ)

dNTPs

An automated thermocycler

Amplification of a DNA Segment

Long Product

Long Product5

5

A repetitive three- step process : Denature--Anneal--Elongate

(94-97oC) (42-55oC) (72oC)


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LP

LP

SP

SP

The LongProduct(LP) acts astemplatefor newsynthesis

Gives rise toShort Product(SP) whose5 and 3 endsare both setby the primerannealingpositions


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Sequentialrounds

In subsequent rounds, theShort Products accumulatein an exponential fashion

SP

SP


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TET

TET

DNA ligation reactionis transformed into

competent cellsand then spread onselective agar plates

Following restriction digestion, thevector and insert are purified byagarose gel electrophoresis


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Analysis of Recombinant Clones: Restriction Enzyme Digestion

Log

10

bp

Distance

DNA fragments stainedwith ethidium bromideand visualized by UVillumination.

1.2% agarose gel castIn 1X TAE buffer

Vector

Clone

2

DNAMar k

er

DNAMar k

er

DNAMark

er

CutEcoRI/P

vuII

Clone

2

Insert

CutEcoRI/P

vuII

CutEcoRI/P

vuII

Vector

[uncu

t]

CutEcoRI/PvuII/

NotI

Vector

Insert

EcoR I Pvu II Not I


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Analysis of Recombinant Clones: Thermal Cycle DNA Sequencing

O

H H

HOH

H

Base

H

PO4

O

H H

HH

H

Base

H

PO4

dNTP

ddNTP


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Genomic Library Construction using Bacteriophage -based Vectors

Genes are arranged into functional groups

The genome contains optional DNA

Insertion and Replacement Vectors

Insert size range < 20 kb

* *

* Cos site

Cos sites incorporated into a plasmid = Cosmid


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Genomic DNA Library Construction Analysis: Colony Hybridization

Nytran orNitrocellulosemembrane

Add an in vitropackaging mix


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Restriction digestion

A large segment ofgenomic DNA or achromosome

A whole genome

Shotgun Sequencing Approaches

Closing a sequencing gap

Note:A genomic map is needed toprovide a guide for sequencingby showing the positionsof genes and other distinctivefeatures.


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Part II. Experimental Problems and Approaches Assigning Genes to Chromosomal Locations

Genetic MappingRFLP and SSLP Analysis

Physical MappingPositional Cloning of a Target GenecDNA synthesis and expression cloningMapping Genes using ESTs

Cloning Large Multigene Families by Degenerate PCR

Cloning of a Target Protein and Physical Mapping

Block II Lecture 1: Recombinant DNA Technology


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Genetic and Physical Mapping of a Gene to a Chromosome

Genetic markers used forchromosomal mapping:

Restriction site variation

Repetitivesequences

Genetic Linkage Analysis

Genetic mapping

enables physicalmapping


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Restriction Fragment Length Polymorphism (RFLPs)

Useful molecular marker loci for chromosomal mapping and diagnosis of humandisease genes

This technique takes advantage of the ability of bacterial restriction enzymes to cut DNAat specific target sequences that exist randomly in the DNA of other organisms.

Generally, the target sites are found at the same position in the DNA of differentindividuals within a population (i.e. the DNA of homologous chromosomes).

Frequently, a specific site is missing because of some silent mutation. The mutationcould be within a gene or a non coding intergenic region.

Genetic Mapping


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If an individual is heterozygous for the presence (+) and absence (+/ -) of a restrictionsite, that locus can be used in mapping. The (+ / -) sites are detected by Southernblot analysis using a probe derived from that region.

Homolog 1

Homolog 2

3 kb

2 kb 1 kbExtent of probe

Southern blotanalysis of thisindividuals DNAwould detect threefragments, 3, 2,and 1kb in length.

Another individual might be homozygous for the long fragment and would showonly a 3 kb band on a Southern blot.

Homolog 1 3 kb

Homolog 2

3 kb

Multiple forms of this region constitute an RFLP

Southern blotanalysis of thisindividuals DNAwould detect one

fragment 3kb inlength.

3kb

2kb

3kb

1kb

Extent of probe


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3 kb

2 kb 1 kb

d

D

In a cross of the two previous individuals, 50% of the progeny would show 3fragments when probed, and the other 50% would show 1 fragment. This

result follows Mendels Law of Equal Segregation, just as a gene would.

Homolog 1

Homolog 2

2kb

3kb

1kb

3kb


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3 kb

2 kb 1 kb

d

D

Hence, an RFLP can be mapped and treated like any other chromosomal site.

Linkage of the heterozygous RFLP to a heterozygous gene with D coupled tothe 1 plus 2 morph. Crossover between these sites would producerecombinant products (D-3, d-2-1).

With this approach, the RFLP locus can be mapped relative to othermolecular markers.

Homolog 1

Homolog 2


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Restriction Fragment

Length Polymorphism(RFLP) Analysis

DNA Fingerprintingused in modern forensics

Suspect

Evidence

Victim


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d

D

Probe binds repetitivesequences

The number of repeated units in a tandem array is variable. Individualsheterozygous for different numbers of tandem repeats can be detected,and the heterozygous site (s) used as a marker (s) for mapping.

This VNTR locus will form two bands on a Southern blot: one long andone short. Similar to an RFLP locus, this heterozygous site can be usedfor genetic mapping.At present, VNTR analysis is rapidly performed using PCR.

Restriction target sites are outside the repetitive array.

The basic unit of the array is indicated by the arrows.

Simple-Sequence Length Polymorphisms (SSLPs)

VNTRs :Variation in theNumber ofTandem Repeatsor Mini-satelliteMolecular Markers


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Genetic profiling using Mini-Satellite VNTRs

VNTRs located on the short arm of Chromosome 6 were amplified by PCR.The PCR Products were labeled with a blue or green fluorescent marker andresolved on a polyacrylamide gel. Each lane displays the genetic profile of adifferent individual. No two individuals will have the same genetic profilebecause each person had a different set of mini-satellite variants, which giverise to bands of different sizes after PCR. The red bands are DNA markers.


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Positional cloning of a human target gene

Chromosomal Walking technique used to identify single-disease genes in humans

Contigs


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Isolate mRNA from

cell or tissue ofinterest

Check integrity ofRNA prep on HCHO

gel

Convert total pool

of mRNA into cDNAusing RT

cDNA Synthesis

Clone cDNA into a DNA vector (e.g. Zap) l to construct a cDNAexpression library. Propagate and amplify cDNA library in asuitable host. Screen for cDNA of interest using

DNA probe or antibodies that recognize the encoded protein.

DNA molecules copied froman mRNA molecule by RT

and therefore lack introns ingenomic DNA


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Gene Mapping using Expressed Sequence Tags (ESTs)

EST DATABASEA collection of partial cDNA sequences, generally 200 to 400 bp in length, that was generatedby sequencing vast numbers of cDNAs isolated from human cells and important model organisms

such as mouse, Drosophila, and Caenorhabditis elegans.

Composed of relatively short portions (tags) of genomic DNA sequences that are expressed in theform of mRNA. The EST database is constantly updated as sequences from increasing number ofcDNA clones are added.

cDNA

5

3

ESTs are obtained by sequencinginto the cDNA insert using a primerbased on the vector sequence


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Genetic code contains redundancies = Degenerate

ATT-IleTAT- TyrTTA - Leu

STOP CodonsTAATAGTGA

TTG- LeuCTT- LeuCTC- LeuCTG- Leu

20 Different Naturally Occurring Amino Acids

64 CODONS : 61 encode amino acids


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Computer programs applythe triplet-based genetic codeto translate the ESTsequences into partial aminoacid sequence.Three nucleotides (a codon)

are read from a specificstarting point. If a match isfound, then the EST providesthe unique DNA sequence ofthat portion of the cDNA.

A single probe that iscomplementary to theportion of the EST can beused to screen a genomicDNA library;the probe could also beused to screen a cDNA

library

tyr phe ile ser ser asn ser thr leu asn ala lys leu his leu thrCOOHNH2


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Cloning aLarge

Multi-GeneFamily byDegeneratePCR

Odorant Receptors and the Organization of the Olfactory System


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Experimental design based on three assumptions:

1. Odorant receptors likely belong to a superfamily of receptors

(i.e. seven transmembrane domain receptors) that transduce intracellularsignals by coupling to GTP-binding proteins

2. The large number of structurally distinct odorous molecules suggeststhat the odorant receptors themselves should exhibit significant diversityand are likely to be encoded by a multigene family.

3. Expression of odorant receptors should be restricted to the olfactory epithelium.


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GOAL: To identify molecules in the olfactory epithelium that resemble members of the seventransmembrane domain superfamily.

Step 1. Extract RNA from olfactory epithelium and prepare cDNA

Step 2. cDNA is amplified by PCR using a series of degenerate oligonucleotide primersthat anneal to conserved regions of members of the superfamily of G-coupled seventransmembrane domain receptor genes.

II VII

5 primers (match Domain II sequences)

3 primers (match Domain VII sequences)

Each of the five different 5 primer was used in PCRReactions with each of six different 3 primers.


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Step 4. PCR products within the size range expected for this family of receptor (600-1300 bp) were selectedfor further amplification with the appropriate primer pair to isolate individual bands. Each of the semi-purified PCRproducts was digested with the restriction enzyme Hinfl and analyzed by gel electrophoresis.

(22 of the 64 PCR

products isolated)

PCR 13 yields a very large number of restriction fragmentswhose molecular weight sums to a value 5- to 10-fold greater

than the original PCR product (13 different species of DNA)

Step 3. The amplification products of each PCR reaction were analyzed by agarose gel electrophoresis


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Step 5. PCR 13 DNA was cloned into the plasmid vector Bluescript and 5 clones analyzed by DNA sequencing

Each clone exhibited a different DNA sequence,BUT each encoded a protein that displayed

conserved features of the superfamily of seventransmembrane receptor proteins.

The proteins encoded by all 5 clones shareddistinctive sequence motifs not found in othersuperfamily members , indicating they were allmembers of a NEW family of receptors

Step 6. Obtain full-length cDNA clones by screening cDNA libraries prepared from olfactory epithelium RNAor RNA from enriched populations of olfactory sensory neurons

Primary screen used a mixture of PCR 13 DNA as the probe (20 positives)Secondary screen used the original pair of primers used to amplify PCR 13 DNA (A4/B6)

Step 7. Confirm expression of isolated cDNAs is restricted to epithelium using Northern blot analysis

RESULT:Identified 18 members of a novel, extremely large multi-gene family that encoded olfactoryreceptors and lead to future work that merited the 2004 Nobel Prizein Medicine.


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Cloning of a target protein X

Protein X Encoded by a pathogen Gene locus unassigned

COOH

NH2

Digest with protease

COOH

H2 N

H2 N

COOH

COOHH2 N

COOH

H2 N

COOH

H2 N

Separatepeptides

Automated peptidesequencing

Step 1

Step 2

Step 3

D i d b b d i l i


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Design a degenerate probe based on partial protein sequence

Once DNA sequence of the target gene is available, you could: Map the entire gene and its location within the pathogen genome Clone and sequence the transcript(s) encoded by the Protein X gene Define the Protein X gene structure Construct expression plasmids for functional studies of Protein X in cells Mutagenize the Protein X cDNA using PCR-based site-directed mutagenesis andperform structure-function analysis

Produce recombinant protein X for vaccine development studies

Mixture of 96 oligonucleotidesthat encode a portion of the peptide

Degenerate

PCR

Note: If a Protein X EST database existed, you could design a single probe that

was based on partial protein sequences


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REFERENCE MATERIALS FOR BLOCK 2/ LECTURE 1/ DNA Manipulations

Lehninger Principles of Biochemistry, 3rd edition, Chapter 29

An Introduction to Genetic Analysis , 7th edition, Chapters 6, 7, 12, and 13

(http://WWW.WHFREEMAN.COM/BIOLOGY)

FYILab Math: A handbook of Measurements, Calculations, and OtherQuantitative Skills for Use at the Bench. D.S. Adams (2003) CSH

Laboratory Press.
http://www.whfreeman.com/BIOLOGYhttp://www.whfreeman.com/BIOLOGY

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