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University of Aden
Dr. Adeel Altuhish
Academic year 2018-2019
Polymerase Chain Reaction
(PCR)
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
• 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
PCR components
1-Target DNA
2-Primer
3- nucleotides
4-enzymes
1- DNA template
• DNA containing
region to be
sequenced
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.
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.
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)
I-5- 12
Annealing
 Hybridize
 Primers anneal to denatured template DNA
 Tm of primers
 Annealing temperature
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.
RT-PCR
• Reverse Transcriptase PCR
• Uses RNA as the initial template
• Yields ds cDNA.
• Reverse transcriptases are used
by retroviruses to replicate their genomes,
Detection of amplification
products
• Gel electrophoresis
• Sequencing of amplified fragment
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)
Advantages
• fast, reliable (reproducible) results
• less chances of contamination)
• High output
• Broad uses
• Defined, easy to follow protocols
References
• Fundamentals of Biochem ( Voet, Voet,
Pratt)
• Molecular Cell Biology ( Lodish, Darnell..)

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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
  • 5. 1- DNA template • DNA containing region to be sequenced
  • 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)
  • 9.
  • 10.
  • 11.
  • 12. I-5- 12 Annealing  Hybridize  Primers anneal to denatured template DNA  Tm of primers  Annealing temperature
  • 13.
  • 14.
  • 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,
  • 19.
  • 20.
  • 21.
  • 22. Detection of amplification products • Gel electrophoresis • Sequencing of amplified fragment
  • 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
  • 25. References • Fundamentals of Biochem ( Voet, Voet, Pratt) • Molecular Cell Biology ( Lodish, Darnell..)