Methods in reproduction Lecture One Flashcards
What are two methods of amplifying DNA?
1) PCR
2) Cloning
What does PCR achieve?
- Selectively Amplifying DNA to be Analysed
What is the rate of DNA amplification in PCR?
Logarithmic - so doubles ever cycle
How does PCR amplify DNA?
In vitro amplification using enzymatic processes.
What does the latest generation of PCR involve?
- Computerised thermal cylinders, which automatically heat and cool during a cycle
- thermostable Taq polymerase isolated from Thermus aquatics
What are the steps of PCR?
1) Denaturation
2) Annealing
3) DNA synthesis
What happens in denaturation?
The system is heated to 90-95 degrees celsius and the two strands of DNA seperate
What happens in the annealing stage of PCR?
A primer and reverse primer attach to the single strand of DNA and form brackets around the specific sequence
Around 50-65 degrees
What happens in the DNA synthesis stage of PCR?
Tay Polymerase attaches and produces a complimentary strand of DNA (72 degrees)
Are the temperatures consistent for all PCR?
No- it depends on the enzyme being used
Describe PCR in simple terms:
PCR is the process of selectively amplifying DNA by denaturation (separation of DNA strands), annealing primers and using Taq Polymerase to produce the target complimentary sequence.(DNA amplification)
This is a logarithmic reaction in a thermal cylinder.
What are the applications of PCR?
- Substitute for cloning
- To amplify specific regions of DNA from small amounts of material
- Selectively detect foreign DNA sequences in tissue being tested i.e viruses
- Analysis of highly degraded DNA
What are the ingredients for PCR?
- Target DNA
- Specific primers
- Deoxyribosenucleoside triphosphate (bases)
- Taq DNA polymerase
- PCR buffer
NO DNA LIGASE
What are some characteristics of a good primer?
- Must be specific
- 18-30 base pairs
- melting temp (56-75 C)
- GC content 40-60%
- 3’ end, ending with G or C
- Avoid secondary structures
- Avoid runs of bases
- Avoid intraprimer homology (primers adhering to one another)
How can the products of PCR be prepared for analyses?
Gel electrophoresis
How does gel electrophoresis work?
It separates DNA fragments according to size and conformation
What are the types of gels and their uses in gel electrophoresis?
Polyacrylamide gel - single strand DNA less than 500bp
Agarose gel - 300-20,000 bp
Pulsed-Field gel - Long DNA molecules
Whats special about agarose gel?
DNA moves from negative to positive terminals when a current is applied.
How is a agarose gel read?
The separated DNA is stained with ethidium bromide and exposed to UV, becoming visible and reference well indicates the various mass of DNA (as short fragments move faster and therefore further within the allocated time.
What would you do if you had non-specific product?
Increase the annealing temperature
What would you do if you had no product?
Decrease the annealing temperature
When would you alter the magnesium concentration?
When no product was present (post-lab find out what this is)
When would you alter the amounts of primer?
When there is a concentration imbalance between the two?
When would you increase the number of cycles?
When a small faint product is produced
When would you decrease the number of cycles?
When a large indistinguishable product is produced.
What does DNA sequencing achieve?
Allows you to read the nucleotides of the DNA strand
Whats a method of DNA sequencing?
Replicate a target sequence using normal deoxyribosenucleoside triphosphate and a trace amount of dideoxyribosenucleoside triphosphate (these terminate the sequence) so on gel electrophoresis the fragments are separated on size and there are four troughs, one for each base thats tagged i.e A,G,C,T. So the first nucleotide in the sequence will move the furthest and increasing numbers of base pairs will be behind.
Whats another method of DNA analysis?
automated sequencing using fluorescent primers. Then using a laser to identify the order of bases.
What is NGS?
Next generation sequencing
Whats an example of NGS and why is it good?
Massively parallel DNA sequencing.
- High throughput
- Reduced cost
- Downside is hat massive amounts of sequencing data is produced.
Whats an application of DNA sequencing?
Non-invasive prenatal testing using fetal DNA in the maternal plasma
How is non-invasive prenatal testing achieved?
- Fetal DNA represents 15% of the DNA in maternal plasma.
- Uses massively parallel sequencing of maternal plasma and can detect some detail chromosome aneuploidies (abnormal chromosome number)
- Only those at high risk in NZ are eligible to be screened.
How is NGS used in the lab?
- IN UoA they look to see if ageing oocytes DNA mutates
- Mapping genes to determine what causes disease.
What are some examples of mRNA analysis techniques?
Northern blot in situ hybridisation Reverse transcriptase polymerase chain reaction Real time RT-PCR Microarray analysis of gene expression
Why is RT-PCR used?
Highly sensitive and rapid
What is real time RT-PCR?
The use of a Taq man probe. This primer is designed to break become dislodged and break, becoming fluorescent when the RT reaches it during the DNA replication. The quantity of fluorescence is proportional to the amount of gene present.
What is hybridisation?
Formation of double strand DNA between two single strands that are complimentary and one is labelled.
What are some examples of hybridisation methods?
Dot Blot Southern blot Fluorescence In Situ Hybridisation (FISH) SNP arrays Microarray Array CGH
What does microarray analysis of gene expression have in terms of characteristics?
- Enables simultaneous analysis of thousands of genes
- detection of distinct gene pattern expression.
Do the last five slides of lecture one post lab
do it now
