1. Polymerase Chain Reaction

2. Problem Suppose you have a patient with an
infection or a heritable disease.
You want to know which infection or
disease it is and…..
you want to know it fast and .....
from as little material as possible
Is that feasable?

3. Which DNA technologies are available?
Southern blot
In situ hybridization
Sequencing
PCR

4. What is relevant in medical diagnostics?
SSS
Speed the faster the better
Sensitivity sample size
Specificity reliability of diagnosis

5. Which type of DNA can be used for what. genes
Which type of DNA can be used for what? genes - repeat DNA: centromere DNA telomere DNA CA repeats - junk DNA

6. Which type of DNA can be used for what?
Junk ???
Repeat identification
Gene diagnosis of disease
‘foreign’ DNA detection of infection
CA-repeats turn out to be useful for identification
of individuals. What is a CA repeat? Why?
Mutations in genes can lead to hereditary diseases.
PCR (in combination with sequencing) helps to detect
such mutations.
PCR can amplify non-self DNA assist in detecting
infections of viruses, bacteria or parasites.

7. CA-repeats turn out to be useful for identification of individuals.
What is a CA-repeats
CA-repeats turn out to be useful for identification of individuals.
5’ ’
CACACACACACACA
Primer primer
Fixed location in the genome but the length
Differs among individuals

8. Principal of a PCR assay on CA repeats
CA repeats vary in length from 4 to 40 bp
and can be found on positions
in the genome. Of each CA-repeat
an individual gets one copy of the
father and one copy of the mother.
The number of
primer sets in the
PCR determines
the specificity of
the identification.

9. PCR
PCR, polymerase chain reaction, is an in-vitro technique for amplification of a region of DNA whose sequence is known or which lies between two regions of known sequence
Before PCR, DNA of interest could only be amplified by over-expression in cells and this with limited yield

10. 1966, Thomas Brock discovers Thermus Aquaticus, a thermostable bacteria in the hot springs of Yellowstone National Park
1983, Kary Mullis postulated the concept of PCR
( Nobel Prize in 1993)
1985, Saiki publishes the first application of PCR
( beta-Globin )
1985, Cetus Corp. Scientists isolate Thermostable Taq Polymerase (from T.Aquaticus), which revolutionized PCR

11. Reaction Components
DNA template
Primers
Enzyme
dNTPs
Mg2+
Buffers

12. 1- DNA template DNA containing region to be sequenced
Size of target DNA to be amplified : up to 3 Kb

13. 2- Primers 2 sets of primers Generally 20-30 nucleotides long
Synthetically produced
complimentary to the 3’ ends of target DNA
not complimentary to each other

14. Primers
Not containing inverted repeat sequences to avoid formation of internal structures
40-60% GC content preferred for better annealing
Tm of primers can be calculated to determine annealing T0

15. 3-Enzyme
Usually Taq Polymerase or anyone of the natural or Recombinant thermostable polymerases
Stable at T0 up to 950 C
High processivity
Taq Pol has 5’-3’ exo only, no proofreading

16. The PCR Cycle Comprised of 3 steps:
1. Denaturation of DNA at 950C Primer hybridization ( annealing) at C DNA synthesis ( Primer extension) at 720C

22. Standard thermocycle

23. RT-PCR Reverse Transcriptase PCR Uses RNA as the initial template
RNA-directed DNA polymerase (rTh)
Yields ds cDNA

26. rTth DNA polymerase is a thermostable DNA polymerase derived from the thermophilic bacteria Thermus thermophilus (Tth) HB8.  The enzyme has a reverse transcriptase activity in addition to a 5’→3’ polymerase activity and a double strand specific 5’→ 3’ exonuclease activity in the presence of Mn2+ ions.

28. Detection of amplification products
Gel electrophoresis
Sequencing of amplified fragment
Southern blot
etc...

29. Applications Genome mapping and gene function determination
Biodiversity studies ( e.g. evolution studies)
Diagnostics ( prenatal testing of genetic diseases, early detection of cancer, viral infections...)
Detection of drug resistance genes
Forensic (DNA fingerprinting)

30. Advantages Automated, fast, reliable (reproducible) results
Contained :(less chances of contamination)
High output
Sensitive
Broad uses
Defined, easy to follow protocols

31. In Conclusion
PCR is sensitive and versatile diagnostic tool: 1) to detect hereditary diseases 2) to detect infections 3) to monitor development of a disease 4) to identify fathers or other criminals PCR is extremely useful if one . 1) knows the sequence of a gene. 2) carefully selects primers 3) avoids contamination

32. Instrumentation

33. Real Time PCR

34. END