DNA Replication and PCR Flashcards
DNA molecules can be…
… separated and annealed.
Why can dna strands be easily separated?
because hydrogen bonds are weak
the strands can be easily
separated because hydrogen
bonds are weak - and then be
made to…
…reanneal.
the strands can be easily
separated because hydrogen
bonds are weak - and then be
made to reanneal
this is important in gene
expression because…
… its how genes are transcribed.
the double helix can be ‘melted’ forming what?
2 single stranded DNA polymers.
How are genes transcribed in eukaryotes?
as single units.
In eukaryotes genes are transcribed as single units however in bacteria genes involved in the same
pathways are often arranged linearly as an operon
When the operon is transcribed all of the genes are transcribed in one continuous mRNA known as a
polycistronic mRNA
What does this increase the efficiency of?
This increases the efficiency of gene regulation
– for instance if the bacterial cell needs to make biotin it makes sense to transcribe all of the genes involved in this process together
–means there can be a simple genetic switch to turn the process on.
DNA is polymerised by…
… a complex of proteins at the replication fork.
What is the function of dna polymerase?
catalyses the addition of bases to the 3’ end
Whats the function of Topoisomerase?
unwinds the helix to prevent torsion
Funcition of helicase?
melts the double stranded molecule
Function of DNA primase?
makes short RNA primers (gives a free 3’ end)
Whats the function of single stranded binding proteins?
prevents premature annealing
Whats the function of DNA ligase?
Joins okazaki fragments on the lagging strand.
DNA strands run…
…antiparallel
Each strand of the double helix actually is antiparallel to its…
… complementary strand.
Each strand of the double helix actually is antiparallel to its complementary strand.
Why does this come about?
Because DNA is polymerised only in one direction (5’ to 3’). Nucleotides are added at the 3’ end. This is where DNA polymerase joins, and generate from 5’ to 3’.
The 5 and 3 here refer to the…
carbon atoms on the deoxyribose sugar that joins with phosphate groups.
DNA polymerase needs small ‘primers’ – short sequences of RNA (made by RNA polymerase) to…
…start it off.
In order to synthesise new strands of DNA several things are needed:
1) A complementary strand of DNA (template DNA)
2) An excess of short oligonucleotide primers to provide a free 3’ end
3) Molecules of the 4 bases: dATP, dCTP, dGTP, and dTTP
4) A dna polymerase enzyme (Taq polymerase - thermostable)
5) Magnesium ions (Mg2+) - a co-factor for all DNA polymerases.
6) Buffer to maintain pH to correct value for DNA polymerase.
How can we amplify dna?
By using the previously described elements and cycling between denaturing, annealing and extension temperatures we can amplify DNA from a few molecules to many molecules.
Describe dna amplification?
Starting with very few copies after each round of amplification there is a theoretical doubling of the number of DNA molecules
After only 30 cycles of denaturing, annealing and extension 1 original
molecule will result in…
… 1 billion DNA molecules.
Describe step 1 of PCR?
1) Denaturation
By heating the DNA to 95°C we can denature the double strand because the weak hydrogen bonds are broken, so the two strands are separated.
Describe step 2 of PCR?
2) Annealing
If we have in excess lots of primer molecules, then those primers will anneal to their complementary sequence.
Describe step 3 of PCR
3) Extension
If we now add in a polymerase and the nucleotides dATP, dCTP, dGTP, dTTP then the complementary strand will
polymerise.
What needs to be done in order to understand how a multicellular organism works?
we need to identify which genes are turned on at
which tissues at certain times.
Understanding how a multicellular organism actually
works we need to identify which genes are turned on at
which tissues at certain times.
In this example we may want to examine gene expression
from mouse embryos at different developmental stages –
What will this give us an insight into…
which genes are important at different stages of development (genes that show
temporal regulation)
How do we look at whether a gene has been turned on or off in an organism?
1) by examining RNA levels
2) by examining protein levels.
When examining RNA levels or when examining protein levels, what are looking at?
the gene product to see whether a gene has been turned on or off.
To study gene expression, what technique can we use?
RT-PCR (reverse transcription-PCR)
RT-PCR (reverse transcription-PCR) involves…
… the formation of complementary DNA (cDNA) to mRNA in the cell, remember mRNA has a poly-A
tail.
Why does RT-PCR (reverse transcription-PCR) work?
the technique works because:
1 - single stranded DNA will base pair to single stranded RNA
2 – the enzyme reverse transcriptase can polymerase DNA using an RNA
template
a complementary DNA strand (cDNA) is produced after the addition of…
… a poly-T primer
mRNA has a…
… poly-A-tail
after first strand synthesis of reverse transcription, we are left with…
… a ssDNA molecule (RNA is digested)
Once we are left with our ssDNA, if we now add two primers and Taq polymerase we can…
… do a PCR amplification
how is cDNA formed?
cDNA is formed from mRNA, reverse transcriptase, and a poly-T-primer
What’s the purpose of a “no DNA template” or “No Template Control” lane in PCR?
A No Template Control (NTC) lane in PCR experiments ensures that there is no contamination in the reagents or in the environment. The NTC lane consists of all the PCR components but with no DNA template. If there is any amplification in this lane, there has been contamination. This could lead to false-positive results. Having this lane ensures the PCR results are accurate.
Purpose of a positive control in PCR?
Ensures that the PCR is capable of giving a positive result. This is done using a sample which is known to give a certain result. If the positive control gives its expected outcome, researchers know the PCR is working correctly. If it does not produce this expected result, they know the setup is not functioning correctly and there is a problem.
What is the purpose of genomic dna lane in pcr?
Used as a reference or control to make sure pcr setup is working correctly. Contains genomic dna where the target dna sequence is from. Researchers can compare the results from the genomic dna lane and the lane of the target DNA sequence to ensure the pcr is working correctly and efficiently.
purpose of doing a pcr of actin?
The purpose of performing a PCR of actin, specifically beta-actin, is to use it as a loading control. Beta-actin is a housekeeping gene that is consistently expressed at stable levels across different cell types and conditions. This makes it an ideal reference to ensure that the amount of sample loaded in each lane is consistent. By comparing the expression of beta-actin to the target gene, researchers can normalize the data and account for any variations in sample loading or experimental conditions
What is one major problem of RT-PCR?
one major problem of RT-PCR is that it is only semi-quantitative.
in the PCR reaction, once many
molecules of amplified product have been made they begin to…
…compete with primers during the annealing step – so less product is made
How does RT-PCR work?
the mechanics of PCR combined with reverse transcription – various
enzymes, buffer, dNTPs, primers – 3 step PCR process after RT, then gel electrophoresis.
Why does RT-PCR work?
because DNA forms double
stranded molecules by
complementary base pairing – Taq polymerises new strands but needs a template etc.
Why would we use RT-PCR?
in order to study where or when a gene is being expressed – gene regulation
What does RT-PCR tell us?
qualitative or semi-quantitative
patterns of gene expression
What does RT-PCR NOT tell us?
real quantitative data of
expression – also whether the
gene gets translated to protein – or how active the protein is.
What other techniques other than RT-PCR would give us more information?
qPCR, northern blotting,
transcriptomics, studies of the
protein itself
What is the solution to RT-PCR’s major problem?
qPCR
Rules of designing primers?
- obviously the sequence has to be complementary
- length of primer – usually around 20-25 bases (the longer it is the higher the
annealing temperature and the more specific it is) - at least 50% of the bases should be G or C (because they have more hydrogen
bonds – they increase the annealing temperature) - there should not be repeats within the bases
- the primers shouldn’t be complementary to each other
- if possible the 3’ (last) base should be a G or C
- the melting temperatures (Tm)of the two primers should be similar
To express the protein, you need to design primers to the …
… ‘Open reading frame’ (contains no stop codons).
