DNA analysis Flashcards
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
what is the purpose of glycerol?
glycerol is a dense substance which helps the DNA samples sink into the wells
what are the stains used for visualising colourless DNA bands?
- ethidium bromide and UV light
- methylene blue
how does ethidium bromide and UV light work to stain the DNA bands?
EtBr is added to the agarose gel before it is poured into the gel casting tray
After the DNA fragments are separated by gel E, the agarose gel is placed under UV light to visualize the bands
how does methylene blue work to stain the DNA bands?
After the DNA fragments are separated by gel E, the entire agarose gel is soaked in methylene blue overnight
=> methylene blue binds to DNA and each DNA band will be visible as blue bands
how can the products of gel E be used to estimate the size of DNA molecule?
compare the position of the DNA bands with the DNA ladder to estimate the length of the DNA molecules
how can products of gel E be used to analyse PCR products?
- the thickness of DNA bands can indicate the amount of DNA amplified
- the length/size of the amplified DNA can be checked by comparing the DNA bands with the DNA ladder
how can products of gel E allow for isolation and purification of DNA sample?
DNA can be obtained from agarose gel after gel E is complete, provides a way to isolate pure samples of individual DNA fragments
how can gel E be used to compare and identify different alleles of a gene from different DNA samples?
- obtain DNA samples from each individual and load them into separate wells
- carry out gel E
- different alleles/mutations suggests a difference in nucleotide sequences which can affect restrictions sites obtained when cut with the same restriction enzyme
- the restriction fragments from each allele produce different DNA banding patterns when separated by gel E
define nucleic acid hybridisation (southern blot)
a procedure in which DNA fragments separated by gel electrophoresis are transferred from the agarose gel to a nitrocellulose membrane, the separated DNA fragments are then denatured and hybridised to a radioactive probe
how useful is southern botting when analysing DNA from the entire genome of the organism?
a restriction enzyme digest on the entire genome would result in a large number DNA fragments and a smear instead of discrete bands will appear on the agarose gel during the visualisation of the bands
=> southern blot allows us to visualise only the discrete bands containing the DNA sequences of interest
