1. MISS NUR SHALENA SOFIAN
CHAPTER 6 POLYMERASE CHAIN REACTION - INTRODUCTION - PROCESSES INVOLVED - APPLICATIONS - DNA FINGERPRINTING - R-T PCR
MISS NUR SHALENA SOFIAN

2. INTRODUCTION TO PCR
PCR was invented in 1984 by ( Kary Mullis ) & he received the Nobel Prize in chemistry in 1993, for his invention
It revolutionized biological methods specially in molecular cloning in a way that it has became an inseparable & irreplaceable part of molecular investigations.

3. PCR: What is it?
Polymerase chain reaction: a means of amplifying relatively short pieces of DNA
Advantages:
- very small amounts of DNA
- Wide array of sample
- Ease of extraction
- specifically target particular DNA sequences of varying length

4. The PCR Process: Components of the Reaction
Template DNA
Primers
dNTPs – building blocks of DNA
Thermostable DNA polymerase (Taq polymerase)
Buffer and salts (KCl, MgCl2)
Optional: BSA, DMSO, formamide

5. Thermus aquaticus from hot springs

6. PROCESSES INVOLVE
There are three basic steps which are in common with all type of PCR
1. Thermal denaturation :
In this step DNAs are denatured mostly by temperature about 94˚C & single stranded DNAs are made
( in some cases It’s done by helicase )
2. Primer annealing :
In this step primers are attached to ssDNA by their complementary regions.
3. Extension or polymerization :
This is done by a temperature resistance polymerase named Taq polymerase which is extracted from Thermus aquaticus.

7. DNA REPLICATION PROCESS
Keep the parental strands apart
Breaks the hydrogen bonds between the two strands
Synthesizes daughter DNA strands
III
Alleviates supercoiling
Covalently links DNA fragments together
Synthesizes an RNA primer
DNA REPLICATION PROCESS

11. PCR carries out in thermocycler

12. PCR phases Exponential Linear Plateau
If 100% efficiency – exact doubling of products. Specific and precise
Linear
High variability. Reaction components are being consumed and PCR products are starting to degrade.
Plateau
End-point analysis. The reaction has stopped and if left for long – degradation of PCR products.

13. Contaminations like RNAs must be removed by RNase.

14. Advantages & Disadvantages
The most accurate & feasible technique to determine the amount & concentration of products.
Rapid cycling (30 minutes to 2 hours).
Specific & sensitive.
Not much more expensive.
* * * * *
Pollution.
Poor precision.
Hard to get quantitative data.

15. How are the primers made?

16. Example Target sequence (priming sites are underlined)
5’TATAAGCCATAACGATATTGCTGAGTCAAGTCCACATATCATATGGATGAGAAATGCTTGTGGAGCTGATGTTGATTTGGAGAGACTCTCTCTCTCTCTCTCTCTCTCTAAACCAGTTAAAGAGTGTGCCAGTAGAG3’
Forward primer: 5’ ATG GAT GAG AAA TGC TTG TG 3’
Reverse primer: 5’ACT GGC ACA CTC TTT AAC TGG 3’

17. APPLICATIONS
Amplify a sample of chromosomal DNA – used in DNA fingerprinting analysis
Nonspecific PCR is used to increase total amount of DNA in very small samples e.g. blood stains found at crime scene
To detect and quantitate amount of specific RNAs in living cells – used in RT-PCR
Other applications such as fluorescent labeling, mutation detection

18. DNA FINGERPRINTING IN PCR
Automated DNA fingerprinting whereby a sample DNA is amplified utilizing primers that recognizes end of microsatellites
Microsatellite fragments are fluorescently labeled and separated by gel electrophoresis
The fluorescent molecules excited with laser and measured via fluorescent detector

19. Reverse Transcriptase PCR
One of the most useful applied molecular genetics method
RNA needs to be purified and stored at low temperature to prevent degradation
Two steps:
1. Biochemical separation of total RNA from DNA and protein using protein denaturant
2. Isolation of poly-A and mRNA using oligo dT affinity matrix

20. Reverse Transcriptase PCR
First strand cDNA synthesis
mRNa copied into cDNA by reverse transcriptase using oligo dT primers
Second strand cDNA synthesis
Requires artificial primer to initiate synthesis
Addition of small amounts of RNaseH resulted in production of short RNA primers – promoting DNA synthesis at multiple sites along cDNA template

21. The reaction
PCR mix – Taq polymerase, specific primers, dNTPs and buffers
cDNA is denatured >900C; temperature lowers to C to allow annealing of primers (600 bp apart)
Temperature rises to 720C and Taq polymerase extends the DNA from the primers
The cycle is repeated
The reaction products of cDNA are anaylzed by agarose gel electrophoresis
EtBr is insensitive and does not allow accurate quantitation of cDNA. Alternative is to use SYBR green dye (more fluorescent)
SYBR green binds to ds DNA (reverse transcriptase PCR) and ss DNA (real-time PCR)

22. RT-PCR can be used for cloning
Restriction enzyme sites can be added to the cDNA of interest
Able to generate sticky ends for ligation into vector of choice
2 sticky ends permits directional cloning

23. SYBR GREEN

24. Real-Time PCR
The reverse transcriptase has an endo H activity which removes the mRNA allowing second strand of DNA to be formed
Real time PCR is developed to:
- quantitate differences in mRNA expression
- availability of small amounts of mRNA e.g. cells obtained by laser capture micro-dissection, small amounts of tissue, primary cells, precious reagents
To quantitate mRNA:
- Northern blotting
- Ribonuclease protection assays (RPA)
- in situ hybridization
- PCR

25. Real-Time chemistries allow for the detection of PCR amplification during
the early phases of the reaction. Measuring the kinetics of the reaction in
the early phases of PCR provides a distinct advantage over traditional
PCR detection.

26. Real Time PCR - Diagnosis
Quantitatively measurement of Human Immunodeficiency Virus (HIV).
Detection of Thalassemia, hemophilia, sickle cell anemia & fauvism by real time PCR.
Cystic fibrosis.
Phenyl ketonuria.
Use in forensic medicine.
Non-invasive Prenatal Diagnosis by Analysis of Fetal DNA in Maternal Plasma.
Detection and Quantitation of Circulating Plasmodium falciparum DNA.
Effect of antimicrobial peptides on host cells

27. Conclusion
PCR has proved to be a useful tool in research and diagnosis.
Its use has also brought new challenges to research in terms of interpreting the results due to sensitivity and quantitative measurement
In medicine, PCR-based diagnostics are just becoming widely used and because of the increased cost-effectiveness of the newer assays, knowledge for their interpretation will soon become available