DNA Cloning and PCRDNA Cloning and PCR

46 chromosomes 2 copies of a gene (or DNA sequence

of interest) 6 x 109 base pairs ~ 6 pg (6 x 10-12 g)

Beta-globin gene is 0.00005% of the entire genome Dystrophin (2.5 Mb) is 0.08% of the genome

The Diploid Human The Diploid Human GenomeGenome

General approaches for studying General approaches for studying specific DNA sequencesspecific DNA sequences

DNA CloningDNA Cloning

Goal:Generate large amounts of pure DNA that can be manipulated and studied using a variety of different techniques.

The first major breakthrough for cell based DNA cloning was the

discovery of

RESTRICTION RESTRICTION ENDONUCLEASESENDONUCLEASES

“the Molecular Scissors”

the 1978 Nobel Prize for Physiology or Medicine was awarded to Daniel Nathans, Werner Arber, and Hamilton Smith for their discovery of REs

RESTRICTION ENDONUCLEASESRESTRICTION ENDONUCLEASES

RECOGNIZE and CUT specific 4 – 6 bp PALINDROME sequences known as restriction sites

5’- A G C T - 3’3’- T C G A - 5’

5’- G A A T T C – 3’3’- C T T A A G – 5’

AluI

EcoRI

•Most restriction enzymes occur naturally in bacteria.

•Protect bacteria against viruses by cutting up viral DNA.

•Bacteria protects their own DNA from being cut up by methylation of restriction sites.

•More than 400 restriction enzymes have been isolated and are commercially available.

RESTRICTION ENDONUCLEASESRESTRICTION ENDONUCLEASES

RESTRICTION RESTRICTION ENDONUCLEASESENDONUCLEASES

A G C T

T C G A

AluI

A G C T

T C G A

Blunt ends Sticky ends

G A A T T C

C T T A A G

EcoRI

G A A T T C

C T T A A G

Sticky ends are useful for DNA cloningSticky ends are useful for DNA cloning

DNA Cloning: the stepsDNA Cloning: the steps• Isolate DNA from organism

• Cut DNA with restriction enzymes

• Ligate each piece of DNA into a cloning vector cut with the same restriction enzyme to create a recombinant DNA molecule.

• Transform recombinant DNA (cloning vector + DNA fragment) into a host that will replicate and transfer copies to progeny.

Insertion of foreign DNA into a Insertion of foreign DNA into a VectorVector

CLONING VECTORCLONING VECTOR

A small DNA molecule into which a another DNA fragment of an appropriate size can be integrated1.Can replicate independently of a host cell chromosome2.Produces many identical copies of the inserted gene3.Carries at least one gene that gives it a selectable trait

Plasmid vectors have the Plasmid vectors have the following featuresfollowing features

• Origin sequence (ori) required for replication.

• Selectable trait that enables E. coli that carry the plasmid to be separated from E. coli that do not (e.g., antibiotic resistance).

• Multiple cloning sites i.e., a large number of restriction sites in a small space

• Simple marker that allows you to distinguish plasmids that contain inserts from those that do not (e.g., lacZ+ gene)

A Plasmid VectorA Plasmid Vector

Clone Selection using Blue/White Clone Selection using Blue/White screeningscreening

Bacterial lacZ gene (-galactosidase)

-galactosidase hydrolyzes a bond in a dye called X-gal, turning it blue

The cloning site for foreign DNA is in the lacZ gene

DNA inserted = -galactosidase inactive = White bacterial colonies in the presence of X-gal

DNA not inserted = -galactosidase active = Blue bacterial colonies in the presence of X-gal

TransformationTransformation

The process whereby new DNA (such as a plasmid) is incorporated into a bacterial host

• Treating bacteria with CaCl2• Heat shock bacteria at 42oC followed by

placing on ice• Treating bacteria in a electric current

(electroporation)

Possible outcomes of a Possible outcomes of a cloning experimentcloning experiment

Bacterium does not take up a plasmid

Bacterium takes up a non-recombinant plasmid

Bacterium takes up a recombinant plasmid

Grow bacteria on medium Grow bacteria on medium that contains ampicillin and that contains ampicillin and

X-galX-gal

Recombinant DNA Recombinant DNA technologytechnology

Recombinant DNA technologyRecombinant DNA technology

• Recombinant proteins• Transgenic plants (Genetically

modified crops)• Transgenic animals• DNA vaccines

Protein Expression Protein Expression VectorsVectors

• Should be able to be transcribed and translated by the genetic machinery of the bacteria into which it is introduced– Promoter for RNA polymerase– Ribosomal binding site– Transcription terminator sequence

A Protein Expression vectorA Protein Expression vector

Polymerase Chain ReactionPolymerase Chain Reaction

Polymerase Chain Reaction Polymerase Chain Reaction In vitroIn vitro DNA cloning DNA cloning

It is the SELECTIVE AMPLIFICATIONSELECTIVE AMPLIFICATION of a single specific DNA sequence from within a heterogeneous mixture of DNA (usually whole genomic DNA)

Basic requirements of DNA Basic requirements of DNA replicationreplication

PCR is DNA replication in a test tube

• A DNA template• Primers• Nucleotides• DNA polymerase

• MgCl2• Buffer

Prior information is required about the DNA sequence flanking the target sequence

TARGET SEQUENCE

FLANKING SEQUENCE

FLANKING SEQUENCE

• Two primers are required for each PCR reaction, complimentary to opposite strands with their 3’ ends pointing towards each other

PCR primersPCR primers

Properties of PCR Properties of PCR primersprimers

• Specific for the sequences flanking the target sequence

• Optimally 18-25 nucleotides long• No self complimentary regions within

the primer OR regions of complimentary sequences between the two primers

PCR- the basic processPCR- the basic processSeries of cycles of three successive steps (30

sec – 1 min)

• DENATURATION OF DNA– At 95oC

• ANNEALING OF PRIMERS– From 50-70oC

• EXTENSION OF TEMPLATE(DNA synthesis)– At 72oC

30-35 cycles

PCR was revolutionized by isolating DNA PCR was revolutionized by isolating DNA polymerase from bacteria (polymerase from bacteria (Thermus Thermus

aquateusaquateus) that live in hot water springs) that live in hot water springs

DNA increases exponentially in DNA increases exponentially in each cycleeach cycle

The DNA of interest is amplified by a power of 2 for each PCR cycle

6 cycles of PCR = 25 or 64 copies of DNA 40 cycles of PCR = 240 or 1,099,511,627,776

or 1.099 x 1012 copies of DNA!!!

DNA increases exponentially in DNA increases exponentially in each cycleeach cycle

PCRPCR

Forward primer & Reverse primer +

Template DNA +

dNTPs+MgCl2+Taq Polymerase

2-3hrs2-3hrsAMPLIFIED PCR PRODUCT

PCR Product on an agarose gel

Advantages of PCRAdvantages of PCR• Rapid and easy to perform• Sensitive, amplification of DNA from

minute samples is possible• Robust, making it possible to amplify DNA

from degraded samples.

Disadvantage of PCRDisadvantage of PCR• Prior sequence knowledge• Short size range of amplification products

– 100 bp - 5000 bp• Chances of contamination