1. DNA Cloning and PCR

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

3. General approaches for studying specific DNA sequences

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

5. RESTRICTION ENDONUCLEASES “the Molecular Scissors”
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

6. 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

7. 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.

8. RESTRICTION ENDONUCLEASES
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

9. Sticky ends are useful for DNA cloning

10. 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.

11. Insertion of foreign DNA into a Vector

12. 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

13. 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)

14. A Plasmid Vector

15. 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

16. 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)

17. 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

18. Grow bacteria on medium that contains ampicillin and X-gal

19. Recombinant DNA technology

20. Recombinant DNA technology
Recombinant proteins
Transgenic plants (Genetically modified crops)
Transgenic animals
DNA vaccines

21. 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

22. The average human genomic DNA library consists of ~75,000 independent clones

23. cDNA Libraries
For the analysis of protein coding regions i.e., exons

24. 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

25. A Protein Expression vector

27. Polymerase Chain Reaction

28. 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)

29. Basic requirements of DNA replication
PCR is DNA replication in a test tube
A DNA template
Primers
Nucleotides
DNA polymerase
MgCl2
Buffer

30. Prior information is required about the DNA sequence flanking the target sequence
FLANKING SEQUENCE
TARGET SEQUENCE
FLANKING SEQUENCE

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

32. Properties of PCR primers
Specific for the sequences flanking the target sequence
Optimally nucleotides long
No self complimentary regions within the primer OR regions of complimentary sequences between the two primers

33. 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

34. PCR was revolutionized by isolating DNA polymerase from bacteria (Thermus aquateus) that live in hot water springs

35. DNA increases exponentially in each cycle

36. 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, or x 1012 copies of DNA!!!

37. PCR Template DNA + Forward primer & Reverse primer +
dNTPs+MgCl2+Taq Polymerase
2-3hrs
AMPLIFIED PCR PRODUCT

38. PCR Product on an agarose gel

39. Advantages of PCR Disadvantage 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 bp
Chances of contamination