PCR Flashcards
1
Q
Minimum requirement for PCR in vitro
A
- a strand of DNA to act as a template
- a short, single strand of DNA complementary to part of the template (the primer)
- DNA polymerase
- dNTPs
- Mg+
2
Q
PCR
A
- invented by Kary Mullins in 1983, published in 1986
- Mullis’ insight: enzymatic copying of double stranded DNA using 2 primers, completmentary to opposite strands would lead to exponential increase in amount of target sequence
- led to Nobel prize and japan prize in 1993
3
Q
PCR process
A
- requires DNA to be cycled repeatedly through 3 temperatures
- generally 30-35 cycles (allowing for more than 1million fold amplification
4
Q
Denaturation
A
- temp: 94-96 degrees C
- double stranded DNA denatures into single stranded DNA
5
Q
Annealing
A
- temp: 50-65 degrees c (dependent on melting temp of primers)
- primers bind to complementary sequences
6
Q
Elongation
A
- temp: 72 degrees
- DNA polymerase binds to annealed primers and extends DNA at 3’ end of the chain
7
Q
PCR ingredients
A
- dNTPs
- Mg2+ (in form of MgCl2) essential for enzyme, affects primer annealing
- primers
- template DNA (needs to be pure… can be double stranded or single stranded)
- thermostable DNA polymerase (Taq)
- original PCR required addition of DNA polymerase after every denaturation step)
- a salt
- pH control (Tris)
- stabilizers
8
Q
PCR
A
- primers=short molecules of single stranded DNA (oligonucleotides) most often 16-25 b long
- can be shorter or longer
- priming between two oligos annealed to opposite strands can give exponential growth of product
- PCR products up to 40kb have been produced but most involves 2kb or less
- yield drops with increasing length of DNA product
9
Q
Principles of primer designs (1)
A
-primers should usually be 18-25 b long
-below 18b there is a greater chance the primer will bind to DNA off target
-above 25b, little gain in specificity and primers become more expensive
-probability of random match between primer and DNA template = 0.25^n where n is the number of bases in the primer
-
10
Q
Principles of primer design (2)
A
- 3’ end of the primer is most crucial
- need an exact match at this position
- at least first 3b should be complementary
11
Q
Principles of primer design (3&4)
A
- one or two mismatches between primer and DNA may be ok as long as it is not at the 3’ end
- it is ok to add sequences of arbitrary length at 5’ end of primer that don’t match the template
12
Q
Principles of primer design (5-7)
A
- Base composition should be reasonably balanced so that the melting temperature is similar for primers
- Melting temp = (4x(G+C))+(2x(A+T))
- Primers should have complementary 3’ ends
13
Q
Applications of PCR
A
- Amplifying target sequence for further study
- amplifying a target sequence from within a complex mixture is equivalent to purifying the sequence of interest
- must know enough about the sequence of interest to design effective primers
- detection of rare DNA sequences: can detect as little as a single copy of DNA sequence
- eg: detection of: bacterial contaminants in food, bacteria in environmental samples, pathogens in organisms, forensics, environmental DNA
14
Q
Stages of PCR
A
- during early cycles of PCR, production of DNA product is only limited by the amount in the previous cycle —> exponential growth of product-in later cycles, dNTPs are less abundant and DNA polymerase may start to wear out leading to slower growth of product —> linear phase
- eventually growth in amount of PCR product slows down greatly and then stops as polymerase and dNTPs start to become exhausted
- Cq value marks first point product clearly exceeds detached threshold of instrument
- log-linear phase provides the best information to estimate starting amount of DNA template
15
Q
Real-time PCR
A
- growth in amount of PCR product is monitored by using a reporter dye, and a PCR machine capable of detecting fluorescence in each well
- SYBR green is simplest and cheapest reporter dye
- SYBR fluoresces much more strongly when bound to double stranded DNA
