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 AnalysisThermal Cycle DNA Sequencing

Library Construction and AnalysisShotgun Approaches for Sequencing Genomic DNA

Cloning: To make identical copies

DNA cloning involves separating a specific gene or DNA segmentfrom a chromosome, attaching it to a DNA carrier molecule, and replicating 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 DNA molecules.

The methods used to accomplish these and related tasks are collectively referred to as recombinant DNA technology or genetic engineering.

•Developed from naturally occurring bacterial plasmids

•Contain an origin of replication (ori )

•Contain numerous restriction sites

•Contain genes that confer resistance to antibiotics, thusallowing selection ofbacterial colonies carrying the plasmid

•Introduced into competent bacterial cells by transformation

Cloning vectors allow amplification of inserted DNA fragments

Different types of cloning vectors

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

Bacteriophage-based Cosmids: Linear DNA molecules used for genomic and cDNA cloning. Bacterial host. Insert size range < 20kb.

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

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

Mammalian expression vector

Note : MultipleCloning Sites (MCS)or a Polylinker Region

With the exceptionof budding yeast,plasmids are uncommonin eukaryotes.Thus, most eukaryoticvectors are based on DNA or RNA viral genomes.

*

*

**

*

* * Viral DNA sequences

Bacterial sequences

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

Restriction endonucleases cut DNA molecules at defined positions

Blunt and sticky ends

5’ and 3’ overhangs

The same sticky ends produced by different enzymes

Digestion of DNA with different restriction endonucleases

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 whichflank the sequence of interest

A DNA Template (a few ng)

A thermal-stable DNA 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)

Polymerase Chain Reaction (PCR)

LP

LP

SP

SP

The Long Product(LP) acts as templatefor new synthesis

Gives rise toShort Product(SP) whose 5’ and 3’ endsare both setby the primerannealing positions

Polymerase Chain Reaction (PCR)

Seq

uen

tial

ro

un

ds

In subsequent rounds, theShort Products accumulatein an exponential fashion

SP

SP

TE

T

TE

T

DNA ligation reaction is transformed into“competent cells”and then spread on selective agar plates

Following restriction digestion, thevector and insert are purified by agarose gel electrophoresis

Analysis of Recombinant Clones: Restriction Enzyme DigestionL

og

10 b

p

Distance

DNA fragments stainedwith ethidium bromideand visualized by UV illumination.

1.2% agarose gel cast In 1X TAE buffer

Vec

tor

Clo

ne

2

DN

A M

arke

r

DN

A M

arke

r

DN

A M

arke

r

Cu

t E

coR

I / P

vu II

Clo

ne

2

Inse

rt

Cu

t E

coR

I / P

vu II

Cu

t E

coR

I / P

vu II

Vec

tor

[ u

ncu

t ]

Cu

t E

coR

I / P

vu II

/ N

ot

I

Vector

Insert

EcoR I Pvu II Not I

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

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

Genomic DNA Library Construction Analysis: Colony Hybridization

Nytran orNitrocellulose membrane

Add an in vitropackaging mix

Restriction digestion

A large segment of genomic DNA or a chromosome

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.

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

RFLP and SSLP Analysis Physical Mapping

Positional 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

Genetic and Physical Mapping of a Gene to a Chromosome

Genetic markers used for chromosomal mapping:

Restriction site variation

Repetitive sequences

Genetic Linkage Analysis

Genetic mapping enables physical mapping

Restriction Fragment Length Polymorphism (RFLPs)

Useful molecular marker loci for chromosomal mapping and diagnosis of human disease genes

This technique takes advantage of the ability of bacterial restriction enzymes to cut DNA at 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 different individuals within a population (i.e. the DNA of homologous chromosomes).

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

Genetic Mapping

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 blot analysis of this individual’s DNA would detect three fragments, 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 23 kb

Multiple forms of this region constitute an RFLP

Southern blot analysis of this individual’s DNA would detect one fragment 3kb in length.

3kb

2kb

3kb

1kb

Extent of probe

3 kb

2 kb 1 kb

d

D

In a cross of the two previous individuals, 50% of the progeny would show 3 fragments when probed, and the other 50% would show 1 fragment. Thisresult follows Mendel’s Law of Equal Segregation, just as a gene would.

Homolog 1

Homolog 2

2kb

3kb

1kb

3kb

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 produce recombinant products (D-3, d-2-1).

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

Homolog 1

Homolog 2

Restriction FragmentLength Polymorphism(RFLP) Analysis

“DNA Fingerprinting”used in modern forensics

Su

spec

t

Evi

den

ce

Vic

tim

d

D

Probe binds repetitive sequences

The number of repeated units in a tandem array is variable. Individuals heterozygous 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 and one short. Similar to an RFLP locus, this heterozygous site can be used for 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 of Tandem Repeatsor “Mini-satellite”Molecular Markers

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.

Positional cloning of a human target gene

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

Contigs

Isolate mRNA from cell or tissue of interest

Check integrity ofRNA prep on HCHO

gel

Convert total pool of mRNA into cDNA using RT

cDNA Synthesis

Clone cDNA into a DNA vector (e.g. Zap) l to construct a cDNA expression library. Propagate and amplify cDNA library in a suitable host. Screen for cDNA of interest usingDNA probe or antibodies that recognize the encoded protein.

DNA molecules copied froman mRNA molecule by RTand therefore lack introns ingenomic DNA

Gene Mapping using Expressed Sequence Tags (ESTs)

EST DATABASEA collection of partial cDNA sequences, generally 200 to 400 bp in length, that was generated by sequencing vast numbers of cDNAs isolated from human cells and important model organismssuch 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 of cDNA clones are added.

cDN

A

5’3’

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

Genetic code contains redundancies = Degenerate

ATT-Ile TAT- Tyr TTA - Leu

STOP Codons TAATAGTGA

TTG- Leu CTT- Leu CTC- Leu CTG- Leu

20 Different Naturally Occurring Amino Acids

64 CODONS : 61 encode amino acids

Computer programs applythe triplet-based genetic code to translate the EST sequences into partial amino acid sequence. Three nucleotides (a codon) are read from a specific starting point. If a match is found, then the EST provides the unique DNA sequence of that portion of the cDNA.

A single probe that is complementary to the portion of the EST can be used to screen a genomic DNA library;the probe could also be used to screen a cDNAlibrary

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

Cloning a Large Multi-Gene Family by Degenerate PCR

Odorant Receptors and the Organization of the Olfactory System

Experimental design based on three assumptions:

1. Odorant receptors likely belong to a superfamily of receptors (i.e. seven transmembrane domain receptors) that transduce intracellular signals by coupling to GTP-binding proteins

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

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

GOAL: To identify molecules in the olfactory epithelium that resemble members of the seven transmembrane 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 primers that anneal to conserved regions of members of the superfamily of G-coupled seven transmembrane 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 PCR Reactions with each of six different 3’ primers.

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 PCR products 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 greaterthan the original PCR product (13 different species of DNA)

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

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 seven transmembrane receptor proteins.

The proteins encoded by all 5 clones shared distinctive sequence motifs not found in othersuperfamily members , indicating they were all members 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 olfactory receptors and lead to future work that merited the 2004 Nobel Prize in Medicine.

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

Separate peptides

Automated peptide sequencing

Step 1

Step 2

Step 3

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 and perform structure-function analysis • Produce recombinant protein X for vaccine development studies

Mixture of 96 oligonucleotides that encode a portion of the peptide

DegeneratePCR

Note: If a Protein X EST database existed, you could design a single probe thatwas based on partial protein sequences

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)

FYI Lab Math: A handbook of Measurements, Calculations, and Other Quantitative Skills for Use at the Bench. D.S. Adams (2003) CSH Laboratory Press.