Alvey - Molecular Biology Techniques Flashcards
name the reagents required for PCR
template (DNA or cDNA) primers (need to know DNA seq for these) enzyme (Taq polymerase) dNTPs Buffer - MgCl2 Appropriate temps - thermocycler
name the stages in a PCR reaction and the temps they occur at
starts at 25°C
DNA denaturation - 95°C
Primer annealing - 50°C
Primer extension - 72°C
how is it ensured that DNA doesn’t reanneal in a PCR reaction?
primers anneal to DNA as they’re added in excess - DNA doesn’t reanneal
name the 2 DNA polymerases that can be used in PCR
Taq polymerase - Thermo aquaticus
PFU - high fidelity and proofreading ability
how can PCR results be analysed (hint: we have done this in labs)
Run gels – look at band size (how much PCR product you have)/specificity (how many bands you have)
name 7 applications of PCR
Genetic screening – modify primers to pick out certain mutations
Pathogen detection – viral DNA in blood sample
DNA fingerprinting - genetic makeup of a organism
Gene expression analysis
Sequencing
Template generation for cloning
Gibson Assembly
what is RT-PCR? why is it used?
reverse transcription - PCR used to find out: how much a certain gene is being expressed where the gene is being expressed splice variants in a population how much mRNA is being produced
what are the steps in RT-PCR?
mRNA + reverse transcriptase + primer = cDNA
RNAse removes RNA – (ss)cDNA left
Add primers and polymerase = dsDNA formed
DNA can now be amplified in PCR
what is q-PCR? why is it used?
Quantitative real-time PCR
Used to measure amount of template
describe the steps in qPCR
1) Denaturation step
2) Annealing step
3) Extension step + SYBR green (fluoresces when attached to dsDNA)
name a specific type of qPCR and explain how it works
Allele-specific PCR:
Specificity comes from primers
SYBR green is non-specific, binds to any dsDNA
Amount of fluorescence proportional to amount of dsDNA produced
At end of extension step is when fluorescence is measured
qPCR is measured in cycles using an arbitrary unit called cycle Threshold (cT)
Measure background level of fluorescence by not adding the template immediately (compare fluorescence readings to this value)
After each extension step the PCR machine measures fluorescence
what does a low cT number indicate in qPCR?
a low cT number = a higher copy number of template DNA (it takes fewer cycles of qPCR to overcome the threshold value of fluorescence)
what is site-directed mutagenesis?
In-vitro mutagenesis technique
Used to introduce specific changes into a DNA sequence (you have to know the original sequence for this) to find out whether an a/a or protein is particularly important in an organism you are studying
which amino acid is used to replace a suspected important a/a in site directed mutagenesis?
alanine - v boring a/a
describe the steps of site-directed mutagenesis
1) Design primers that work in both directions, with one deliberate mismatch in middle of the primer
2) PCR amplifies all around the plasmid
3) Dpn1 digestions of template (dsPlasmids are mainly made up of two newly synthesised strands, however ones that are made up of a new strand and an old strand are digested by endonucleases to remove the mismatched base pair)
4) Transformation of mutant plasmid (added to E. coli - this repairs the nicks in the DNA where the newly synthesised DNA meets the primer)
what is a methylation-dependent restriction enzyme? give and example of one and state why is it used in site-directed mutagenesis
a methylation-dependent restriction enzyme is a restriction enzyme that only digests methylated DNA.
example: DpnI
why is it used?: in a PCR reaction the DNA that is amplified isn’t methylated. Therefore the only methylated DNA present in a sample is the DNA that you started with (which doesn’t contain the mutation you have added with your primers). This means that the methylated DNA doesn’t contain your deliberate mutation, so you want it gone - DpnI degrades this DNA for you
what is sanger sequencing?
essentially a DNA synthesis reaction by the addition of ddNTPs to create fragments increasing in length
Allows DNA sequencing of 1000-1500 base pairs of good quality sequence
Still most accurate sequencing method
what 2 pieces of information do you have to know in order to carry out sanger sequencing?
the length of the fragment and the base at the last position
list the reagents required for sanger sequencing
Template DNA Oligonucleotide primer Buffer (including Mg) dNTP ddNTP (small amount) DNA polymerase (Taq)
how do you analyse the results of sanger sequencing using a polyacrylamide gel?
you put each of your 4 tubes of sanger sequences (ddATP/ddDTP/ddCTP/ddGTP) into 4 separate wells at the top of the gel. at the 5’ end (furthest away from the wells) is your smallest fragment, and at the 3’ end (closest to the wells) is your largest fragment. as you have made a complementary strand to your template strand you have to use complementary-base pairing rules to work out your original sequence
describe an easier way of analysing sanger sequences than using polyacrylamide gels
Fluorescent detection
Each ddNTP is tagged with a fluorescent marker (4 different colours) which a machine picks up and analyses. The last base is determined by the colour of the marker.
Can run the reaction at the same time, instead of four separate reactions.
if in your fluorescent analysis of you sanger sequenced DNA there is a double peak in
a single base position what does this indicate about your DNA sequence?
If in a chromatogram there is a double peak at one base position, then:
- The organism the template is from is a heterozygote
- This means there were 2 templates present in the reaction mix (one from each chromosome)
name 2 limitations of sanger sequencing
Only sequence one template at a time
Need to have some knowledge of the sequence to design the primer
genomes and expression profiles are sequenced using NGS. what is NGS? what are the common features of it?
next generation sequencing
common features:
- Millions of reacts occur in parallel
- These reactions are spatially separated
- No sequence knowledge of the template is necessary (this is achieved by adding adapters to the template in which DNA is synthesised from)
describe the steps in traditional cloning
- Chose restriction enzyme that cuts vector once
- Digest insert and vector with same enzyme (or enzyme that creates complementary sticky ends)
- Ligate insert into vector
- Transform into E. coli to amplify and maintain the plasmid
name 3 limitations of traditional cloning
Restricted to single insertion per cloning cycle
Inefficient for large inserts
Plasmid design can be restricted by restriction site availability (ie enzyme may cut gene in middle)
