Vishram Singh - Textbook of Anatomy Upper Limb and Thorax.. Volume 1 (1).pdf
PCR.Lecture 5.ppt
1. University of Aden
Dr. Adeel Altuhish
Academic year 2018-2019
Polymerase Chain Reaction
(PCR)
2. Introduction
• Polymerase Chain Reaction(PCR):
• The polymerase chain reaction is an artificial
method of replicating DNA under laboratory
conditions
• amplification of a region of DNA whose
sequence is known or which lies between two
regions of known sequence
3. • The PCR technique is used to amplify large
quantities of a specific sequence of DNA from
an initial sample
• Each reaction cycle doubles the amount of DNA
6. 2- Primers
A primer is a short strand of RNA or DNA (generally 20-30
nucleotides long). It is Synthetically produced complimentary to
the 3’ ends of target DNA.
Function :
-Aprimer serves as a starting point for DNA synthesis. It
is required for DNA replication because the enzymes that
catalyze this process.
-allow the polymerase to start, because it can't just start by itself
on a single stranded piece of DNA.
7. 3-DNA polymerase Enzyme:
Usually Taq Polymerase Stable at T0 up to 950 C.
-The DNA polymerase is an enzyme work during
the process of DNA replication.
4- deoxyribose nucleotide triphosphate
(dNTPs) to growing DNA strand.
8. Stages of PCR
• PCR occurs in a thermal cycler and uses variations
in temperature to control the replication process via
three steps:
• Denaturation – DNA sample is heated to separate it
into two single strands (~95ºC for 1 min)
• Annealing – DNA primers attach to the 3’ ends of
the target sequence (~55ºC for 1 min)
• Elongation – A heat-tolerant DNA polymerase
(Taq) binds to the primer and copies the strand
(~72ºC for 2 min)
15. I-5- 15
Elongation
DNA polymerase synthesizes (polymerizes) new
DNA molecule by adding deoxyribonucleoside
complementary to the corresponding template base
in a 5’ to 3’ direction.
16.
17.
18. RT-PCR
• Reverse Transcriptase PCR
• Uses RNA as the initial template
• Yields ds cDNA.
• Reverse transcriptases are used
by retroviruses to replicate their genomes,
23. Applications
• Genome mapping and gene function
determination
• Biodiversity studies ( e.g. evolution studies)
• Diagnostics ( testing of genetic diseases,
early detection of cancer, viral infections...)
• Detection of drug resistance genes
• Forensic (DNA fingerprinting)
24. Advantages
• fast, reliable (reproducible) results
• less chances of contamination)
• High output
• Broad uses
• Defined, easy to follow protocols
