Lecture 4: PCR Flashcards
what are two key applications of PCR
- medical diagnosis
- forensic investigations
what does PCR stand for
polymerase chain reaction
why is PCR useful
allows for the analysis of tiny amounts of genetic material
true/false gene cloning and DNA sequencing require large quantities of high-quality, intact DNA
true
what are the components needed for PCR
- template DNA
- DNA polymerase
- the four deoxynucleoside trisphosphates (dNTPs): ATGC
- two primers
what are the three steps of PCR and what happens
- denaturation solution containing the DNA is heated to separate into 2 strands
- annealing solution is cooled and primers anneal to their complementary sequences
- extension DNA polymerase synthesizes new DNA strands by going from 5’ to 3’
What is typically the size for a PCR template region?
400-500 base pairs
True/false The amplified region is often longer than the template duplex
- false
- the other way around
How long are PCR Primers
20-30 nucleotides in length
What part of the primers are always oriented to each other
the 3’ end of the primer
how much of the primers are added to the mix
The primers are added to the reaction mixture in great excess to ensure pairing with any complementary sequence
True/ False Theoretically there is an upper limit to PCR amplification
- True
- because there is no dlifing clamp the polymerase will fall off eventually
How do you claculate the number of DNA after a certain number of cycles?
of starting DNA molecules x 2^n
what is the melting temp (tm)
the temperature at which half the helical structure is lost
what should Tm for the 2 primers be within of each other
5 degree celsius
what can Tm also be referred to as
Ta (annealing temp)
what is the equation for Tm
Tm = [4(G or C) + 2(T or A)] - 5
true/false the 3’ end match is most important for synthesis
true
true/false the 3’ end can have extra bases
- false
- the 5’ end can have extra
- for example, adding restriction enzyme sites to the primer
what are the 2 types of primerss?
- forward
- reverse
where does the forward primer anneal to
the strand going 3’ to 5’
where does the reverse primer anneal to
the strand going 5’ to 3’
what has to be done to get the reverse primer
take the reerse compliment
what are 3 key factors in PCR popularity
1) Availability of DNA polymerase that are resistant to high temperatures
2) Automation of temperature control heat blocks
3) Drastic decrease in the costs of thermocyclers and heat tolerant DNA polymerases
how did PCR work in the early days
- there were 3 heat blocks or water baths of diff temps
- the tubes were manually transfered for each step
- also new polymerases had to be added cause the e.coli polymerase was destroyed by the high temp during each denaturation step
what polymerase is now commonly used for the process of PCr
Taq Polymerase
what is the key difference between e.coli polymerases and Taq polymerase
- e.coli polymerase is destroyed by high temp in the denaturation
- Taq polymerase comes from a bacterium called thermus aquaticus, which is found in hot springs, so it can tolerate the high temps
what machine is PCR performed in
thermocycler
what is a thermocycler
an electronically controled heat block that is programmed to change automatically to the diff temps needed for each PCR cycle step also means that you don’t have to be physically present for every single step
what happens in gel electrophoresis
- dna samples are inserted into wells on the edge of the gel
- using an electrical current, the DNA fragments move towards the positive pole according ot their size
________ (smaller/ larger) fragments move faster in gel electrophoresis
smaller
________ (smaller/ larger) fragments move slower in gel electrophoresis
larger
what does a band represent in gel electrophoresis
represents the DNA of a particular size
true/false the size and amount of DNA is correlated
- false
- just because you have a 1000 bp fragment doesn’t mean you have more than what is in a 400 bp fragment
what represents the amount of DNA in gel electrophoresis
amount of dna is represented by the brightness of the band
what is used to make the gel in gel electrophoresis
- agarose powder
- TAE buffer
- indicator
true/false In gel electrophoresis size relates to distance traveled
true
what is used in gels to determine the size od the band
Premade ladders with bands of known sizes
what is what is ethidium bromide
an intercalating agent (ie inserts itself between DNA bases)
true/false bands cannot be separated and recovered for later use
- false
- they can!
what does RT PCR stand for
reverse transcriptase polymerase chain reaction
what is the purpose of RT PCR
to isolate total RNA from a sample, remove gDNA, use polymerase that uses an RNA template to make a DNA strand (cDNA)
what does qPCR stand for
quantitative polymerase chain reaction
what is the goal of qPCR
measured the amplification in real time during amplification
true/false enzymes exist that can form RNA directly from RNA templates
false
true/false PCR can be used for diagnostic purposes but not forensic purposes
- false
- can be used for both
how can we use qPCR to determine the starting conc of the mRNA of interest
by monitoring the fluorescence
what is sequence analysis
the comparison and alignment of sequences with control and database sequences
what is used for DNA profiling
short tandem repeats
what happens when you increase the number of regions investigated
- you increase the variability,
- but you end up decreasing the probability of finding another with the same combination of repeats
the length of the stretches in DNA of STR vary depending on what
depending on the number of copies of the repeat found at a specific reegion (locus) of DNA
