Polymerase Chain Reaction Problem Suppose you have a patient with an - - PowerPoint PPT Presentation

▶
polymerase chain reaction problem
SMART_READER_LITE
LIVE PREVIEW

Polymerase Chain Reaction Problem Suppose you have a patient with an - - PowerPoint PPT Presentation

Oct 17, 2022 191 likes •538 views

Polymerase Chain Reaction 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

slide-1
SLIDE 1

Polymerase Chain Reaction

slide-2
SLIDE 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?

slide-3
SLIDE 3

Which DNA technologies are available?

  • Southern blot
  • In situ hybridization
  • Sequencing
  • PCR
slide-4
SLIDE 4

What is relevant in medical diagnostics?

  • SSS
  • Speed the faster the better
  • Sensitivity sample size
  • Specificity reliability of diagnosis
slide-5
SLIDE 5

Which type of DNA can be used for what?

genes

  • repeat DNA: centromere DNA

telomere DNA CA repeats

  • junk DNA
slide-6
SLIDE 6

Which type of DNA can be used for what?

  • Type of DNA:

Junk ??? Repeat identification Gene diagnosis of disease ‘foreign’ DNA detection of infection CA-repeats turn out to be useful for identification

  • f 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.

slide-7
SLIDE 7

What is a CA-repeats

  • CA-repeats turn out to be useful for

identification of individuals.

5’ 3’

  • --------- CACACACACACACA------------

Primer primer Fixed location in the genome but the length Differs among individuals

slide-8
SLIDE 8

Principal of a PCR assay on CA repeats CA repeats vary in length from 4 to 40 bp and can be found on10.000 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.

slide-9
SLIDE 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

slide-10
SLIDE 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

slide-11
SLIDE 11

Reaction Components

  • DNA template
  • Primers
  • Enzyme
  • dNTPs
  • Mg2+
  • Buffers
slide-12
SLIDE 12

1- DNA template

  • DNA containing

region to be sequenced

  • Size of target DNA

to be amplified : up to 3 Kb

slide-13
SLIDE 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

slide-14
SLIDE 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

slide-15
SLIDE 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
slide-16
SLIDE 16

The PCR Cycle

Comprised of 3 steps:

  • 1. Denaturation of DNA at 950C -
  • 2. Primer hybridization ( annealing) at 40-500C
  • 3. DNA synthesis ( Primer extension) at 720C
slide-17
SLIDE 17
slide-18
SLIDE 18
slide-19
SLIDE 19
slide-20
SLIDE 20
slide-21
SLIDE 21
slide-22
SLIDE 22

Standard thermocycle

slide-23
SLIDE 23

RT-PCR

  • Reverse Transcriptase PCR
  • Uses RNA as the initial template
  • RNA-directed DNA polymerase (rTh)
  • Yields ds cDNA
slide-24
SLIDE 24
slide-25
SLIDE 25
slide-26
SLIDE 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.

slide-27
SLIDE 27
slide-28
SLIDE 28

Detection of amplification products

  • Gel electrophoresis
  • Sequencing of amplified fragment
  • Southern blot
  • etc...
slide-29
SLIDE 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)
slide-30
SLIDE 30

Advantages

  • Automated, fast, reliable (reproducible) results
  • Contained :(less chances of contamination)
  • High output
  • Sensitive
  • Broad uses
  • Defined, easy to follow protocols
slide-31
SLIDE 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

slide-32
SLIDE 32

Instrumentation

slide-33
SLIDE 33

Real Time PCR

slide-34
SLIDE 34

END