DNA Cloning and PCR

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DNA Cloning and PCR
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The Diploid Human Genome

• 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

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General approaches for studying specific DNA
sequences
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DNA Cloning

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

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• The first major breakthrough for cell based DNA
  cloning was the discovery of
• RESTRICTION ENDONUCLEASES
• 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
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RESTRICTION 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
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RESTRICTION ENDONUCLEASES

• 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.

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RESTRICTION ENDONUCLEASES
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Sticky ends are useful for DNA cloning
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DNA 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.

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Insertion of foreign DNA into a Vector
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CLONING VECTOR

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

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Plasmid vectors have the following 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)

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A Plasmid Vector
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Clone Selection using Blue/White screening

• Bacterial lacZ gene (b-galactosidase)
• b-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 b-galactosidase inactive White
  bacterial colonies in the presence of X-gal
• DNA not inserted b-galactosidase active Blue
  bacterial colonies in the presence of X-gal

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Transformation

• 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)

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Possible outcomes of a cloning experiment
Bacterium does not take up a plasmid
Bacterium takes up a non-recombinant plasmid
Bacterium takes up a recombinant plasmid
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Grow bacteria on medium that contains ampicillin
and X-gal
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Recombinant DNA technology
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Recombinant DNA technology

• Recombinant proteins
• Transgenic plants (Genetically modified crops)
• Transgenic animals
• DNA vaccines

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Genomic DNA libraries

• A collection of cloned DNA fragments obtained by
• the partial restriction digestion of the total
  DNA of an organism
• ligation into an appropriate vector
• Replication within the host bacteria
• Two types
• genomic DNA libraries
• cDNA libraries

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The average human genomic DNA library consists of
75,000 independent clones
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cDNA Libraries

• For the analysis of protein coding regions i.e.,
  exons

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Protein Expression Vectors

• 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

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A Protein Expression vector
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(No Transcript)
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Polymerase Chain Reaction
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Polymerase Chain Reaction In vitro DNA cloning

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

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Basic requirements of DNA replication

• PCR is DNA replication in a test tube
• A DNA template
• Primers
• Nucleotides
• DNA polymerase
• MgCl2
• Buffer

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Prior information is required about the DNA
sequence flanking the target sequence
FLANKING SEQUENCE
TARGET SEQUENCE
FLANKING SEQUENCE
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PCR primers

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

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Properties of PCR primers

• 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

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PCR- the basic process

• Series 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
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PCR was revolutionized by isolating DNA
polymerase from bacteria (Thermus aquateus) that
live in hot water springs
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DNA increases exponentially in each cycle
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DNA increases exponentially in each 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!!!
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PCR
Template DNA
Forward primer Reverse primer
dNTPsMgCl2Taq Polymerase
2-3hrs
AMPLIFIED PCR PRODUCT
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PCR Product on an agarose gel
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Advantages 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 PCR

• Prior sequence knowledge
• Short size range of amplification products
• 100 bp - 5000 bp
• Chances of contamination