R.O Lecture 2 Flashcards
Give examples of cloning whole genetic material
- Of an individual being (whole genomic material), e.g. Dolly the Sheep
- Of a cell, e.g. E.coli (genetic material copied and passed on
to progeny when cells replicate)
Give an example of partial genetic material
- molecular cloning
-Of a DNA sequence, e.g. one gene or part of a gene, by PCR
(polymerase chain reaction) or by transforming into a cell for replication
Give a quick description of the process of recombinant DNA technology
-Identify genetic material of interest (DNA of interest)
-Produce copies of ‘DNA of interest’
-Insert ‘DNA of interest’ into a vector (e.g. plasmid)
-Introduce vector (containing ‘DNA of interest’) into cell (expression system)
Brief history of Polymerase chain reaction (PCR)
-Kary Mullis (1983 87)
-Investigating sickle cell anemia (SNP) but sample was limiting
-PCR: replication of a specific segment of DNA
-1992: Hoffmann La Roche purchased licence from Cetus.
-1993: Nobel prize in Chemistry
What was Mullis’s invention (PCR) based on?
Based on two principles:
Natural DNA replication in cells
Sanger Sequencing Method
Describe the process of Natural DNA replication in cells
-Base pairing enables existing DNA strands to serve as templates for new complementary strands (A:T and G:C)
-Allows replication of genetic information, which is to be passed on to daughter cells during cell division.
-The enzyme DNA polymerase synthesises complementary DNA strand (needs nucleotides, Mg 2+ ions, other enzymes to separate DNA strands).
-Synthesis starts from complementary primer (Primase makes
complementary RNA primer)
-Replication always proceeds in 5’ to 3’ direction
DNA Polymerase
The enzyme DNA polymerase synthesises complementary DNA
strand (needs nucleotides, Mg 2+ ions, other enzymes to separate DNA strands).
It can only bind and start adding on from the 3’ bend
Where does synthesis begin? (natural dna rep in cells)
Starts from the complementary primer.
What makes the complementary RNA primer
Primase
Describe Sanger Sequencing Method
*invented by Sanger, Smith and Coulson in the mid 1970s
*based on synthesis of a complementary DNA strand by DNA polymerase (from E.coli )
*initiated by a short oligonucleotide primer
(DNA replication in a tube)
Function of primers
*Mullis introduced a second ‘reverse’ primer
*primers FRAME your target sequence (requires prior knowledge of the sequence)
Benefit of PCR
Allows us to make billions of copies of a specific DNA sequence
What does the PCR amplification process involve?
Replication process involves thermal cycling
Name the processes in PCR Amplification
- Denaturation
- Annealing
- Extension/Elongation
Denaturation
94 °C
- Heat briefly to separate DNA strands
Annealing
52-65 °C
- Cool to allow primers to form hydrogen bonds with complementary DNA sequence
- Annealing temperature (Ta) picked based on melting temperature (Tm) of primers
How is the Annealing Temperature determined
Ta~ 5 °C below lowest Tm of primers
Annealing temperature depends on the length and composition of the primers used.
Extention/Elongation
72 °C
*DNA polymerase binds and using primers, extends new sequence
*Each cycle lasts 1- 5 minutes and at the end the targeted DNA sequence
is doubled
*The solution is then heated again starting the next cycle of denaturing, primer binding and DNA synthesis
What is the number of cyles for the thermal cycle
30
Why was Taq polymerase incorporated instead of E.Coli DNA polymerase?
- Changed from using E. Coli DNA polymerase to heat stable Taq polymerase (resistant to denaturation)
- Temperature cycling now performed in single instrument
Name the components for PCR reaction
*PCR Buffer
*dNTPs
*DNA Template
*Forward Primer
*Reverse Primer
*Taq polymerase
*dH20
What type of PCR Buffer is used?
PCR Buffer specific for the polymerase but is usually
Tris based pH buffer (pH 8 8.8;
*most now contain MgCl 2 )
Polymerase is usually added last (immediately before reaction starts)
activity declines the longer it is above 0 C
Describe Taq DNA Polymerase
- Integral to ‘cycling’ of PCR: Need a polymerase that is not denatured during high temperature steps
*Produced by T hermus aq uaticus , a bacteria found in hot springs/hydrothermal vents (thermophile)
*heat stable DNA polymerase; elongates DNA at 72 C; resistant to denaturation step of PCR
*Isolated in 1976, Science ‘Molecule of the Year’ 1989
Name other heat-stable polymerases
Pfu and Vent, isolated from other thermophiles both of these have proof-reading capacity
