molecular bio tech Flashcards
What is Polymerase Chain Reaction? (PCR)
It amplifies DNA from a limited source of DNA so that there is sufficient amount for analysis
What are the components of the PCR?
hint: x5
*Template DNA
➔DNA containing the target sequence
to be amplified
* Primers ➔synthetic single-stranded DNA fragment (20-30 nucleotides long) ➔needed to initiate DNA synthesis by providing a free 3’OH group for Taq polymerase to bind to and extend ➔ 2 different primers are required. Each is complementary to the sequence at 3’end of each single stranded target DNA sequence.
➔ are required in large excess to increase the likelihood of them binding to target DNA sequences (instead of target DNA sequences reannealing) ➔ become part of amplified sequences
- Taq polymerase
➔ thermostable DNA polymerase
which is resistant to denaturation at
high temperature - Deoxyribonucleotides (dNTPs)
➔ substrates for DNA replication made
up of dATP, dTTP, dCTP and dGTP - Buffer
➔ contains cofactor, Mg2+
, for proper DNA polymerase function
What is the 3 step process of PCR?
Denaturation
- Heating to 95C causes the weak hydrogen bonds* between complementary bases of each
strand to break due to increased molecular vibrations; - The denaturation of double-stranded DNA into single-stranded DNA exposes the bases for
complementary base pairing;
Primer Annealing
3. Lower temperature of 64C allows primers* to anneal* specifically to the regions flanking the
target DNA sequence via complementary base pairing*;
4. The primers determine the segment to be amplified and provides a free 3’-OH group for chain extension;
Extension
At the optimum temperature of Taq polymerase* , 72 oC, which performs the synthesis of complementary* DNA strand;
6. Chain extension/ elongation* occurs from 3’ end of primer which provides free 3’ OH required
by polymerase;
What are the advantages of PCR?
- Only a minute amount of DNA* is required to carry out PCR as with each round of PCR, the
number of copies of target DNA is doubled. Thus the number of desired sequence increases
exponentially and there will be sufficient DNA for analysis.** - Use of thermostable* (i.e. resistant to denaturation at high temperatures*) Taq polymerase
allows PCR to be automated so DNA can be amplified very quickly.
What are the limitations of PCR?
- Taq polymerase lacks 3’ to 5’ proofreading ability**. Hence errors occurring early in the PCR
reaction will get compounded* with each subsequent replication cycle. - Knowledge of sequences flanking* (i.e. at the 3’ ends of) the target sequence** is required in
order to design appropriate primers. - Taq polymerase tends to ‘fall off’ the DNA template before chain extension is complete if the
strand is too long. Hence there is a limit to the size*** of DNA fragment (~3kb) to be amplified. - Minute amounts* of contaminant DNA* can be exponentially amplified along with target DNA*
and affect the reliability* of the results.
What is the purpose of agarose gel
Separate DNA based on fragment size**
Describe the steps of agarose gel electrophoresis
- A slab of agarose gel is placed in a buffer solution* contains ions* which allows the conduction of electricity** when the current is turned on
- The DNA sample is mixed with a dense loading dye* containing glycerol* & 2 coloured dyes. Glycerol makes the DNA sample denser than the buffer solution so that the DNA sample can sink to the bottom of the well
- Since DNA is invisible, the dyes colour the DNA sample and will indicate if the DNA has been loaded correctly into the well***
- The 2 coloured dyes thus act as visual markers* which help to monitor the progress of the migration* of the invisible DNA fragments in the gel during electrophoresis
- One dye (corresponds to a 100bp DNA fragment) and often runs ahead of the DNA sample* and gives an indication of when gel electrophoresis must be stopped so that the samples do not run out of the gel. The other dye (corresponds to a 1100bp DNA fragment) and gives an indication of the position of the larger fragments on the gel
- DNA samples are pipetted into the wells* in the gel near the negative electrode*
- A DNA ladder (i.e. DNA molecular weigh markers) which contains DNA fragments of known sizes, is run in one of the lanes and acts as a standard for which to compare fragments of unknown size* in the sample
- Negatively charged DNA* is attracted towards the positive electrode (anode) when subjected to an electric current**
- The agarose gel** matrix made of a meshwork of polymer fibres** which impedes movement* of longer fragments more than shorter fragments. The longer fragments thus migrate more slowly* compared to shorter fragments, leading to a banding pattern observed on the gel
- Before the loading dye reaches the end of the gel, the current is turned off
- To visualize the bands***, the gel can be treated with a a staining dye that binds DNA (e.g. ethidium bromide, a carcinogen) and fluoresces under UV light.
- Thus
a) the fragment size* can be estimated (based on position* of the band relative to bands in the molecular weight marker) and
b) the amount of DNA** can possibly be estimated (based on intensity and thickness* of the band)
What is southern blotting
Tool to detect specific nucleotide sequences within a sample of DNA
Describe the procedures of Southern blotting and nucleic acid hybridisation
(Continued from Gel electrophoresis)
1. Gel slab is placed on top of the sponge and under a nitrocellulose membrane. A stack
of paper towels placed on top of nitrocellulose membrane. These are placed in a tray of
alkaline solution. A heavy weight is placed above the paper towels.
- Absorbent paper towels draw the solution towards themselves and the alkaline solution**
denatures* double-stranded DNA* into single-stranded DNA* - Single stranded DNA on the gel is then drawn upwards** onto the nitrocellulose
membrane* and binds to the membrane* (in exactly the same position as they were in
the gel). - Nitrocellulose membrane is removed and incubated with single-stranded, radioactive*
DNA probe which hybridises* via complementary base pairing* to part of the target
sequence. - The excess unhybridised probes are washed off.
- Autoradiography* is performed placing X-ray film** over membrane. Radioactive regions
exposes (& hence blackens) the film* forming an image that correspond to the bands
that have base-paired with probe.
What are Restriction Fragment Length Polymorphisms (RFLPs)?
RFLPs are unique banding patterns** among individuals when their DNA is digested by restriction enzymes** and separated by gel electrophoresis ( and subjected to Southern hybridisation)
What variations are there in RFLPs
- RFLPs arise due to the polymorphic nature of DNA in the different individuals, there will be variations* in the
1. number/location of restriction sites*
or
2. number of tandemly repeated nucleotide sequences* (e.g. short tandem repeats)
This will result in unique banding pattern*
What type of DNA polymorphism is present in sickle cell anaemia?
- a single nucleotide polymorphism*
- difference in a single base pair* due to a point mutation
- in single cell anaemia, this SNP is within the coding region
e. g. in sickle-cell anaemia, the disease-causing mutation occurs at restriction site for Mst II within the beta-globin gene.
1. In the disease causing allele, the Mst II restriction site is eliminated.
2. In the normal allele, Mst II restriction site is retained.
- However the majority of SNPs* used for RFLP analysis are found in non-coding regions
Describe the use of RFLP analysis in determining the genotype of a person for B-globin gene?
Digest genomic* DNA from both samples with MstII to obtain different restriction fragments will arise. (i.e. cut both DNA samples with the
SAME* restriction enzyme, Mst II)
- Perform gel electrophoresis* (which will produce smears on the gel) followed by southern blotting, probing* and autoradiography*
(which will allow the visualisation of distinct bands).
The same single-stranded radioactive probe* (which is complementary to part of the target sequence) will be used to detect both the
presence of A-type and S-type DNA. Hence the site that the probe binds on the DNA fragments should give different banding patterns on
the autoradiogram
Explain the use of RFLP in DNA fingerprinting
- As no individuals (exception of twins) have the same genome, therefore they will not have the same DNA profile.
- The DNA profile is the unique banding pattern that identifies individuals.
How to carry out DNA fingerprinting?
what are the 2 diff steps
- Restriction digestion** of genomic DNA** by restriction enzymes** ( cut DNA from different individuals with SAME restriction enzyme)
followed by gel electrophoresis to separate DNA fragments*** - Southern blotting** using of nitrocellulose membrane.**
- Nucleic acid hybridisation* using radioactive probes complementary to the STRs
- Visualisation of bands** via autoradiography* using X-ray films**
OR
- PCR using primers that flank target DNA sequence
- Restriction Digest
- Gel electrophoresis
- Bands visualised by staining gel with ethidium bromide
What is meant by a restriction enzyme [3]
- Enzyme that recognizes and bind** a specific* 4-6 base pair DNA sequence* called a
restriction site* as its active site is complementary to the DNA sequence; Note:
Binding must be mentioned. - Enzyme cuts/breaks phosphodiester bonds** on specific positions on both DNA
strands; - Giving rise to either blunt or sticky ends* depending on type of restriction enzyme;
- Used as a defence mechanism by bacteria against bacteriophage by cutting up
foreign DNA*, hence restricts multiplication of viruses;
What are sticky ends
and what are blunt ends?
Sticky ends:
- produced when restriction enzymes leave a staggered cut resulting in single-stranded overhangs/ sticky ends
- short overhangs will form H bonds and anneal by complementary base pairing with complementary singe-stranded stretches on other DNA molecules cleaved on the same site
Blunt ends: produced when restriction enzymes make a simple cut across both strands at a single point
How gel electrophoresis is used to separate fragments of DNA. [5]
- Dense* loading buffer is mixed with DNA sample to help it sink* to the bottom of
wells located nearest the negative electrode/cathode**; - Loading dyes also added to DNA sample to allow visualisation** of progress of
electrophoresis; - Negatively-charged** DNA migrates out of well towards direction of positive
electrode/anode when subjected to an electric field / current; - Meshwork* of agarose polysaccharides impede movement of longer fragments
more*** than shorter fragments - causing them to migrate slower** than shorter fragments and end up nearer to the well**;
- A method of visualization is suggested. Either stain gel with ethidium bromide
followed by visualization under UV light. Or Southern Blot *followed by use
radioactive DNA probe** and autoradiography** with an X-Ray film**;
Q1/ tutorial: With reference to Fig. 1.2, explain the banding pattern of individual C. [4]
C - contains 3 bands
B- contains 1 band
A- contains 2 bands
- C is heterozygous for sickle cell
- Normal and mutant alleles are on the same gene loci on a pair of homologous chromosomes
- Normal allele gives rise to an intermediate and short fragment* upon restriction digestion with MstII and these fragments correspond to the middle and bottommost bands respectively
- Mutant allele* gives rise to a single long fragment** upon restriction digestion with MstII and this corresponds with the uppermost band*
IV6 was also found to suffer from the disease, even though genetic screening using the RFLP
marker in question identified him as being only a carrier. Suggest a possible explanation for
this. [2]
1, During formation of gametes in III4 *;
- Crossing over* could have occurred at a locus between disease allele and RFLP
marker, such that disease allele became linked to RFLP allele 3;
IV6 therefore inherited 2 copies of disease allele.
NB: This is one limitation when studying RFLPs flanking the gene responsible for the disease.
However, such RFLPs are still used in genetic screening because they lie very close to the
gene of interest, and therefore crossover frequencies tend to be very low.
Explain why (QV: 1. As number of repeats in STR increases from 7- 12， temperature needed to separate the double strands increases from 53OC (below 55 OC ) - 63.5OC 2. temperature was increasing more slowly as the number of repeats increased )
- With an increasing number of STRs** there would be an increase in length** of
polynucleotides - means larger number of H-bonds** between bases of the two strands to be broken
- Hence, more heat energy** needed to break more bonds** and separate strands
Outline the process of DNA hybridization that allows the RFLP for a particular gene to be visualised. [5]
- ds DNA is denatured / made single-stranded and by alkaline / NaOH solution and
transferred to a nitrocellulose membrane - exactly the same position as they were in the
gel; - Nitrocellulose membrane incubated with a radioactive probe, that is complementary in
sequence* to part of target sequence / gene. - DNA fragments containing this part of target sequence will hybridise to probe* by
complementary base-pairing**;
4(!!) . After hybridisation, membrane is washed to remove any unhybridised probes. - Using autoradiography**/X-ray film over membrane, banding pattern can be visualised.
(Radioactivity of bound probes exposes film to form an image corresponding to bands that
have base-paired to probe.)
Describe the role of the buffer solution in the gel electrophoresis protocol. [2]
[2] - 2 points
- Buffers contain ions which allows conduction of electric current
- Thus allowing negatively-charged DNA** molecules to move from negative
electrode to positive electrode
TAKE NOTE OF POINT 2
In June 2002, over 500 ivory tusks and 42 000 other ivory items were seized from a ship
arriving in Singapore. Evidence seemed to link this ivory to elephants from Malawi in Africa.
(i) Outline the process of genetic fingerprinting using RFLP that could be used to test this
seized ivory. [4]
- Genomic DNA is extracted* from soft tissue/dried blood from seized samples and
Malawian elephants, and cut with same restriction enzyme**; - DNA is separated according to size in gel electrophoresis where negatively-charged DNA** migrates towards positive electrode/anode when subjected to an
electric field / current; - Meshwork of agarose impedes movement of longer fragments more than shorter
fragments* resulting in longer fragments migrating slower than shorter fragments* and
end up nearer to the well*; - ds DNA is denatured* by alkaline / NaOH solution and
transferred to a nitrocellulose membrane*; - Carry out Southern blotting/nucleic acid hybridization by incubating membrane
with single strand radioactive probe* which will hybridise with DNA fragment
through complementary base pairing***; - Using autoradiography/X-ray film*** over the membrane, the banding pattern can visualised
