Fergus - Components of PCR Flashcards
List the six components of a PCR
DNA Template
Primer oligonucleotides
dNTP mix
Thermostable DNA polymerase
Buffer
Additives
Why is a thermostable DNA polymerase needed?
Must be able to denature the DNA (into single strands) without destroying the polymerase activity
Why do we need a buffer and additives?
The thermostable DNA polymerase is used to working in certain conditions e.g. in an organisms where there is a certain pH and certain nutrients available
Give two examples of additives needed for Taq Polymerase
Salt
Magnesium
What type of DNA template are we going to use?
Normally its human DNA but it can often be bacterial or viral (or even viral RNA - has to be converted)
The sequence must contain the target of the amplification
The sequence must not be degraded
Give seven sources of DNA
Peripheral blood
Mouth swab
Tissue sample
Single cell
Cell culture
Archival material
Forensic material
Write a note on using peripheral blood as a DNA source
Mostly used in hospitals
Write a note on using mouth swabs as a DNA source
Not often done only really by ancestry companies - cheap
Write a note on using tissue samples as a DNA source
This can be done for tumours to find out if a mutation has caused the cancer and if the cancer has a therapy
Write a note on using single cells as a DNA source
(3)
Complicated but becoming more common
Needs to be carried out in a really clean environment
Can be done as part of IVF as part of gene therapy i.e. if a family carriers a mutation and don’t want to pass it on to a child they can decide to go through IVF genetic testing so to only grow the embryo without the mutation (a cell is taken off the embryo and the testing is carried out on it, if no mutation is present than the embryo can be implanted)
Write a note on using cell culture as a DNA source
This is mostly used in research e.g. carrying out genetic testing to identify a bacteria etc
Write a note on using Archival material as a DNA source
(2)
Used as controls or examples e.g. tumour blocks
Tumours are often kept after removal and stored, next generation can decide to pursue investigation of mutations by carrying out genetic testing on this sample if there is an inherited condition in the family
Write a note on using forensic material as a DNA source
Using sources of DNA from a crime scene to carry out genetic testing on to identify suspects
Give some examples of DNA templates
(5)
Bacterial DNA
Viral DNA
Vector DNA
cDNA/Genomic Libraries
Products of reverse transcription reactions
What are reverse transcription reactions?
These are carried out on RNA
RNA is reverse conscribed to bring it back to DNA
We can then carry out the PCR on it
Write a note on primer oligonucleotides
(6)
Synthetic DNA sequences
Complimentary to target sequence
Provide primer site for DNA polymerase
Generally 18-30 bp in length
Should not anneal to each other
Should anneal to complimentary sequences at approximately the same temperature
What can we use to pick our primers for us instead of doing it by hand?
We can use software such as primer 3
What is the equation for working out annealing temperature?
(2(A+T) + 4(G+C)) Degrees Celsius
How well should the oligonucleotides match up?
(2)
The 3’ (OH) end should exactly match the target template
The 5’ end can have some modifications
What is a dNTP Mix
(3)
Deoxynucleotide triphosphate builds nucleic acids
There are four types of dNTP, one for each nitrogenous base, in a mix the four are found in varying quantities
Contains dATP, dCTP, dGTP, dTTP
What different materials can be incorporated with dNTP?
(3)
Nucleotide analogues (drugs)
Radioactive nucleotides e.g. 35S and 32P
Fluorescent labels
Give two examples of radioactive nucleotides
32P
35S
Give three examples of fluorescent labels
Fluorescein
Biotin
Digoxigenin
How do you determine the optimum Mg++ concentration for polymerases?
This must be figured out experimentally - trial and error
