Lecture 3 Flashcards
What does PCR stand for?
Polymerase Chain Reaction
What is PCR - the essentials?
It is an in vitro form of DNA replication. It is used to amplify target DNA, producing many copies of the DNA sequence
What is used during PCR?
Specific primers(to allow only the amplification of a specific region of DNA), heat resistant DNA polymerase and nucleotides
What is the aim of PCR?
- To create unlimited DNA copies from low abundant or low numbered DNA
- To amplify the DNA of an impure sample
What are some of the uses of PCR?
- Molecular cloning
- Genome sequencing
- Archaeology
- Forensics
- Clinical Pathology
- Genetic diagnosis
- Characterising unknot mutations
- Finger printing analysis
- Population analysis
- Phylogenetics
How many primers does PCR require?
It requires 2. One that is complementary to each strand of DNA
What is not used in PCR that is used in typical DNA replication?
No need for PCR to use Helicase to unwind the DNA, heating of the sample breaks the bonds that hold together the 2 DNA strands
What are some of the properties of DNA that PCR exploits?
- DNA can be denatured to separate the double strands
- DNA can be annealed both to itself or to appropriately designed DNA fragments
- DNA can be extended using appropriate DNA polymerases
What is the denaturation of DNA and how does it happen?
- The separation of the DNA double strand into its 2 complementary single strands through heating (breaking the H bonds holding the nucleotides together)
- It is carried out at aroun 94-95 degrees Celsius
- Is reversible but dependant on the temperature
What is annealing and how does it happen?
- SIngle stranded DNA allowed to cool in the presence of DNA primers, which are complementary to the start and finish of the region that we want to amplify
- Primers recognise the target sequence of the DNA and bind there
- Typically at 45-60 degrees Celsius (dependant on the primer used)
What is extension and how does it happen?
- DNA polymerase attaches to a free 3’ end on each of the primers and starts to add dNTPs (deoxynucleotide triphosphate) to synthesise a new DNA strand in the 5’ to 3’ direction
- Uses a special thermostable DNA polymerase taken from bacteria that survived in hot springs (optimum temperature works at around 72-74 degrees Celsius)
What happens with each cycle of PCR that happens?
The number of DNA strand doubles every time one PCR cycle happens, this is called exponential doubling
What needs to be put into the PCR micro tube in order to start the PCR process?
- Template DNA (the DNA that is wanting to be copied from) [need less that a microgram)
- Primers (2) forward and reverse primers (targets to tell the reaction where to start and where to stop)
- Thermostable DNA polymers (taq polymerase)
- Deoxynucleotide triphosphates (each of the dNTPs, dATP, dCTP, dGTP and dTTP) [ALWAYS EQUAL AMOUNTS OF EACH OF THE DNTPS)
- Buffer (which contains HCl -stabilising the pH [8-9] KCl -neutralises charge on the DNA and aids with annealing and (NH4)2SO4 - stability, increases the specificity of the binding of the primer and aids in preventing mismatch of primers and DNA
- MgCl2 - Isn’t always needed but can increase efficiency by increasing the activity of the taq enzyme
- Bovine serum albumin - improves the yield of the PCR and controls salt concentration and pH
What is the total reaction volume that is used for PCR?
Between 25-100 microlitres
What are some of the advantages of PCR?
- Very sensitive
- Very quick (produces millions of copies in a few hours)
- Targets a known sequence, doesn’t amplify the whole DNA sequence
What are some of the disadvantages of PCR?
- You already need to know the DNA sequence information to be able to design the primers that will enable the PCR to happen
- Can only amplify quite a small sequence of around 40 kilobases
- Taq polymerase doesn’t ‘proof read’ the bases it has added and so can lead to ‘mismatch’ or point mutations
- The sensitivity will amplify any contamination that is mixed in with the sample (bad especially when used in forensics)
What is colony PCR used for specifically?
It is used amplify bacterial or viral products, mainly used after a transformation event (insertion of DNA into into a plasmid) to check if the DNA has been inserted properly into the plasmid
Why is colony PCR called what it is?
Because a colony off of a culture plate is taken and added in to the PCR mixture
What gene is used if you want to amplify the whole of a bacterial DNA?
16S rRNA gene
What is multiplex PCR used for?
Used to amplify several sections of DNA simultaneously without having to do multiple reactions. There has to be multiple primers (carefully designed, have to make sure they can all work together) to target all regions of DNA that want to be amplified.
What is Quantitative/Real time PCR used for (qPCR)?
Used to determine the amount of DNA in a sample, before amplification started.
Monitors output in real time after each cycle of PCR, it doesn’t use agarose gel electrophoresis it uses probes and dyes that bind to the double stranded DNA (e.g SYBR green)
The fluorescence of the dyes is detected via a laser
What is a negative control and why are negative controls used in PCR?
- It is a reaction mixture that contains everything apart from the template DNA sample (replaced with sterile water)
- It confirms that the target DNA is the only thing that is being amplified and it can detect any contamination that is present, it also allows you to check for any errors in your protocol
What is a positive control and why are positive controls used?
- They confirm the efficiency of the method being used
- It contains all of the normal reaction mixture plus DNA known to contain the target DNA
- You need to know how many copies of the DNA sequence the control contains
If we want to investigate RNA in PCR what has to be done to make this possible?
-It must be copied into cDNA (complimentary DNA) because RNA is not stable at high temperatures [This happens using a primer and a reverse transcriptase enzyme]
How do we usually analyse PCR results?
Agarose gel electrophoresis
How is qPCR analysed?
Through comparison of results against plasmid standards that have been diluted in a dilution series
