DNA analysis Flashcards
(34 cards)
define Polymerase Chain Reaction (PCR)
a technique to amplify a specific DNA sequence without the use of a living organism
=>involves 3 steps: denaturation, annealing of primers and extension of primer
what is the number of strands of DNA produced after PCR?
2^n+1 where n is the number of PCR cycles
what are the materials required for PCR?
- Taq DNA polymerase
- DNA template to be amplified
- forward and backward primers flanking the regions of DNA to be amplified
- 4 deoxyribonucleotides (dATP, dTTP, dCTP, dGTP)
- buffer solution containing MG2+ ions to stabilise the Taq DNA polymerase for optimum enzymatic activity
what are the special characteristics of Taq DNA Polymerase which distinguish it from human DNA polymerase?
thermostable (does not denature at high temperatures)
=> optimum temperature of 72 degrees
has many cysteine amino acid residues, allowing for numerous strong disulfide bonds to be formed
what are the limitations of Taq DNA Polymerase?
lacks 3’ to 5’ exonuclease proofreading ability
=> low replication accuracy resulting in a relatively high error rate
why can PCR only be performed if part of the nucleotide sequence of the DNA template is known?
to allow for the design of the DNA primers
what are primers used in PCR?
short, synthetic, single-stranded DNA fragments
what are the functions of DNA primers?
define the ends of the DNA target sequence to be amplified by
annealling to both strands of the DNA target sequence (complementary to DNA sequences flanking DNA target sequences)
DNA primers are extended towards each other by the addition of deoxyribonucleotides to the 3’ ends of primers
outline the denaturation process of PCR
DNA mixture is heated to about 95 degrees for 60 seconds
>causing the double-stranded DNA to separate
>hydrogen bonds between complementary bases break forming single-stranded DNA
outline the process of annealing of DNA primers of PCR
the DNA mixture is cooled to about 55 degrees for 60 seconds
>to allow complementary primers to anneal to 3’ ends of ssDNA
>excess primers added to prevent the ssDNA from re-annealing to each other
outline the process of extension of primers of PCR
the DNA mixture is heated to 72 degrees for 120 seconds
>Taq DNA Polymerase binds to the 3’ ends of primers
> it reads the bases in 3’ to 5’ direction and adds deoxyribonucleotides to the 3’ end of DNA primers in the 5’ to 3’ direction via complementary base pairing
>new complementary DNA strands are made from the existing primers creating dsDNA molecules
how long is one cycle of PCR
about 4 minutes (billions of copies can be produced in a few hours)
each time the PCR cycle is repeated, the amount of DNA increases by … times
2 times (doubles=2^2)
what are the advantages of PCR? (five)
- amplifies only desired DNA sequence
- PCR is accurate enough to allow amplification of target DNA sequence
- cell-free method of DNA replication
- very sensitive= desired sequence from a single dna molecule can be amplified to produce an extremely large number of DNA molecules w desired sequence
- fast and easy to use=automated
what are the limitations of PCR?(four)
- only short DNA sequence can be amplified
- DNA target sequence must be known in order to design DNA primers for successful amplification
- if DNA mixture is contaminated with DNA from other sources, amplification of unwanted DNA may also take place
- PCR products made by Taq DNA polymerase cannot be used in the cloning of genes because the error rate of the enzyme is too high
in the design of DNA primers, why is it essential to keep the length between 18-22 deoxyribonucleotides?
if primers are too short, they might anneal to non-target DNA sequences
if primers are too long, the rate of annealing to the DNA target sequence will be lower, affecting PCR efficiency
why is PCR only carried out for 30-40 cycles?
efficieny of DNA amplification decreases in the later cycles
=> reduction in amount of DNA primers, increase in amount of PCR products (probability of primers annealing to ssDNA is lowered)
=> due to the increase in amount of ssDNA, ssDNA has a higher probability of re-annealing to form dsDNA instead of to primers
define gel electrophoresis
a technique to separate nucleic acids based on sizes (the size affects the distance moved through a gel matrix under the influence of an electric field)
why do DNA molecules move towards the positive end of the gel?
DNA molecules are negatively charged due to the presence of negatively charged phosphate groups in the sugar-phosphate backbone
what is the function of agarose gel
it forms a cross-linked matrix
it functions as a ‘molecular sieve’ with tiny pores through which the DNA molecules must move across
what are the units used to measure length of DNA fragments?
basepairs or kilobasepairs
what is the purpose of the electrophoresis buffer?
to maintain pH of agarose gel so that DNA molecules remain negatively charged
to allow electricity to conduct evenly through the agarose gel
what is the function of the DNA ladder?
contains DNA fragments of known lengths
=> used as a reference to estimate the lengths of the DNA fragments undergoing analysis
=>usually added in the 1st lane of the agarose gel
what is the function of the loading dye?
the loading dye colours the DNA making it easier to view the DNA samples while they are being loaded into the wells
=> the loading dye travels through the agarose gel ahead of the smallest DNA fragment to allow for the tracking of the progress of gel E to indicate when to turn off the electrical current
