Pcr And Uses Flashcards
In replication what is needed to start rep on both leading and lagging strand
Rna primer 5’ to 3’
Which polymerase primase adds rna primers to the strands in replication
Polymerase alpha
Which polymerase adds dntps in replication to both lagging and leading
Polymerase delta
What does polymerase epsilon do
Removes rna primers
What does ligase do
Joins strands with phosphodiester bonds
What are the SSBs called in replication
Replication protein A (rpa)
Wht holds dna during replication
Sliding clamps
Name all the things needed in PCR
Ssdna template Taq Dntps Buffer - 8.0 and salt Mg or Cl determine primer stringency Balanced temp between primers
Where is the reverse and forward primer
Reverse is at the end of the 5’ to 3’ strand
Forward is at the front of 3’ to 5’ strand
Why do primers help taq polymerase
Needs 3’ oh from the sugar to join to a phosphate
How do you calculate how many dna copies there are
2 to the power of cycle numbers
What is used to detect PCR products in agarose gel
Intercalating dyes (in between bp) They fluoresce under uv light
Give an example of intercalatint dyes
Ethidium bromide (br)
Name the 4 ways PCR is useful
Genetic tests
Diagnosis
Forensics
Personalised medication/ pharmacogenetics
What is qpcr
Monitors the production of and amp of dna every 7 seconds via dye/probe
Why is sybr green used in qpcr
Fluoresces only when dsdna is produced
What is the Ct value
Cycle threshold - the cycle number where a product is detected by qpcr
What is reverse transcriptase rt PCR used for
Measuring levels of gene expression (rna) and viral infections
What is the first step of rt PCR
Rna is converted to cdna strand for PCR to happen
What primer does reverse transcriptase use to convert rna to cDNA
DT primer which is T rich and binds to the poly A tail of mrna on the 3’ end
Why is PCR used to manipulate dna
Understand gene function via deletion/Hr
Protein localisation
What kind of primer is used when transforming cerevisiae budding yeast with PCR products
Hybrid primer
Has bases from yeast dna on the ends and the plasmid dna with the required gene in the middle
How do yeast incorporate the PCR product into their genome
The yeast dna on ends is recognised as broken ends
This causes homologous recombination with the yeast dna and thus incorporates the middle PCR gene from a plasmid origin
What gene is usually transformed into yeast from PCR via the homologous recombination
G418 resistance
How is PCR used to localise proteins
Amplify gfp or other reporter proteins
What does genotyping patients using PCR mean
Identifying which alleles they have for genes
Why is fenotyping patients important
Identify carriers
Pharmacogenetics
HLA tissue typing
Name the 2 techniques used to genotype patients using PCR
PCR rflp
Arms PCR
How is PCR rflp used
Uses RE to cleave the amplified alleles
Eg
If patient has allele 1 which is mutated with an snp, this PCR product will be cleaved and runs further on agarose gel
How is PCR rflp used for sorsbys fundus dystrophy diagnosis
Sorsbys fundus dystrophy is a mutation in timp3 gene which introduces a stop codon prematurely (RE site)
If the PCR product runs faster on agarose gel, they have an allele for sorsbys fundus dystrophy
What is the limitation of PCR rflp
RE are expensive
Only works on known RE sites
How does arms PCR detect allele base variations / mutations
Via allele specific primers
How does arms PCR work
2 types of primers are produced 1 for allele 1 without a mutation or a primer with a known mutation in it
If the PCR product is amplified with a mutated primer, this means the person carries an allele for the mutation
What disease is arms PCR used in
The f508 deletion mutation in CFTR
Which arms PCR uses non specific allele primers
Tetra arms PCR
Why is it important to genotype pathogens via PCR
Detect strain specifically
Better personalised medicine
Why is PCR better at genotyping pathogens than microscopy
Microscopy needs a large sample of pathogen, PCR is sensitive to 1 dna molecule
Microscopy is hard to distinguish strains, PCR is specific
Why is PCR preferred over culturing for genotyping pathogens
Not every strain can be cultured and it takes weeks
What is the advantage of using PCR to genotype pathogens over an antibody response measurement
Not all pathogens produce a antibody response
What would a stronger line on agarose suggest
Bigger infection
What 3 things does PCR tell you about pathogen
If it has a resistance mutation
What strain it is
The presence of it
How is PCR used to phenotype the disease (measure progression)
Measures levels of rna via reverse transcriptase PCR and qpcr is used
What would a low Ct suggest about the phenotype of the disease
It is progressed more, more rna is present as the product is seen faster
