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PCR:

Explain why reverse transcription of mRNA was carried out prior to the PCR reaction. [2]
- Create cDNA from the mRNA in the cell
- Taq polymerase can only act on a DNA template

Explain why DNA polymerase enzyme needed for PCR is derived from a prokaryote that lives in hot
springs, where temperatures may exceed 90oC.
- Enzymes from thermophilic prokaryotes is thermostable/ would not denature at high temperatures
- Thus no need to replenish after each cycle/ allowing process to be fully automated

Explain why DNA polymerase derived from bacterial cells is able to increase the amount of DNA from any
species of organism. [2]
- Most species of organisms share the same genetic code
- Containing adenine, thymine, guanine and cytosine
- Thus active site of DNA polymerase is complementary in shape and charge to DNA nucleotides
from any organism
- To catalyse the formation of phosphodiester bonds between adjacent nucleotides
- To form complementary strands where A=T, C≡G

PROCEDURE
1] Denaturation:
- Hydrogen bonds between DNA strands are broken → DNA is denatured
- By heating to 95oC
- DNA double helix unwinds and separated into 2 separate single strands, each used as a template
for replication by DNA polymerase
2] Annealing:
- Specific DNA primers anneal to single stranded DNA at the 3’ ends
- Via complementary base-pairing
- By cooling to 54oC
3] Elongation:
- Taq polymerase extends primers by adding deoxyribonucleotides to the 3’-OH ends
- By heating to 72oC (optimal temperature of Taq pol.)

ADVANTAGES- Amplification Sensitivity Specificity Robustness Automation Cell-free method Ease of set-up
1. Amplification possible from trace amounts of template DNA
- Target DNA is doubled after every round of PCR/ 2n
2. Easy to set up a PCR and the use of thermocycler
- Commercial synthesis of DNA primers
3. Sensitivity of PCR
- Capable of amplifying sequences from minute amounts of target DNA
4. Specificity in amplifying only target sequences
- As primers hydrogen-bond only to complementary sequences
5. Robustness/ Broad range of DNA sources suitable template for PCR amplification
- Permit amplification of specific sequences from material in which DNA is badly degraded or
embedded in a medium
6. Cell-free method of DNA replication
- Requires no cleanup of unwanted cellular debris or vector DNA
7. Automation of PCR
- Using thermostable Taq polymerase which can withstand high temperature without being
denatured
DISADVANTAGES CPK
1. Highly sensitive to contamination by foreign/non-target nucleic acids
- Hence non-target sequences can be amplified*
2. Taq polymerase used in PCR lacks proof-reading/ 3’ to 5’ exonuclease activity
- Resulting in inaccuracy of DNA replication in vitro
3. Taq polymerase tends to ‘fall off’ the DNA template before chain extension is complete if
the strand is too long
- Thus there is a limit to the size of DNA fragments to be amplified
4. Prior knowledge on the DNA sequence is required for the PCR
- Designing specific oligonucleotide primers is required for the selective amplification of a particular
DNA sequence
5. PCR products labile/unstable as they are not associated with histone*

**The high sensitivity of PCRs makes the method prone to contamination, giving false or inaccurate
results. Carry-over contaminants (e.g. due to aerosolization) from previous PCRs are considered to be
the major source of false positive results. HOW PROBLEM IS SOLVED/REDUCED:
Completely replace dTTP with dUTP during PCR amplification, thereby producing DNA
containing uracil. Prior to initiating PCR, the PCR mixture is treated with Uracil-DNA
Glycosylase (UNG).

Suggest why the graph plateaus.


- Nucleotides are used up, hence unable to make complementary chains
- Primers are used up
- The template DNA strands anneal with each other instead of with the primers
- Taq DNA polymerase gradually becomes denatured ref. no elongation
(*that’s why PCR uses excess dNTP and primers- increase likelihood of them binding to target DNA sequences)

Further research in the 1980s identified sequences of genetic markers located both upstream and
downstream of the gene suspected to cause cystic fibrosis. Using this information, suggest how the gene
could be isolated using these 2 genetic markers. [2]
- Design primers that are complementary to the genetic markers upstream and downstream of the
suspected gene.
- Use the polymerase chain reaction (PCR) to synthesize multiple copies of suspected gene.

Explain how the specific nucleotide sequence of the primer is important for its role in PCR. [2]
- Allows for selective amplification
- Of a segment of DNA
- Via complementary base pairing
- To the 3’ ends of the segments of interests

About 20% of the DNA molecules produced by the PCR are copied inaccurately. Explain why it is not safe
to use PCR to clone the normal CFTR gene for use in treating cystic fibrosis.
- Percentage risk is too high for human application
- Incorrect mRNA formed changes sequence of amino acid in protein
- Produces a non-functional/harmful protein
- Chloride ions not transported/ thick mucus results

HDA is faster than PCR. Explain why.


- No need for heat and cooling cycles thus saves time
- OR Primer annealing and extension steps can occur simultaneously

Explain why the DNA needs to be treated with alkali.


- to denature DNA such that it becomes single stranded
- Breaks hydrogen bonds between 2 complementary strands in order for single stranded probe to
anneal

Explain the role of DNA ladder.


- as a reference/standard
- To estimate the size of unknown DNA molecules

RFLP

Describe the characteristics of a probe.


- a probe is radioactively labelled
- Short stranded DNA
- Which is complementary to target DNA sequence

Besides a variation in number of tandem repeats, name a type of polymorphism that generates different
sizes of restriction fragments.
- single nucleotide polymorphism

Which restriction enzyme would be more suitable to analyze this VNTR in the human population? Explain
your answer.
- EcoRI
- Cleaving with EcoRI would lead to 2 different sized fragments such that the restriction fragments
generated can distinguish the individuals

Suggest why sequences for DNA probes for the cow CHS locus were based on DNA sequences from
humans.
- human DNA sequences are very similar to cow sequences, hence can bind specifically to target
sequences
- homologous gene is found in both cows and humans (NOT identical DNA seq.)

Explain why genes especially protein encoding genes are seldom used as markers for genetic
fingerprinting.
- sequence of protein encoded genes are conserved
- Due to its important biological function
- Mutation rate lower than that in non coding regions
- Not able to differentiate between individuals

Explain the use of genetic markers in genomic mapping in term of linkage mapping.
- genetic markers are scattered throughout each of the chromosomes
- Used to determine the position of the genes
- Frequency with which two genetic markers are inherited together
- Is a measure of the closeness of the two loci on a chromosome
- Recombination frequency indicates the distance between two lock on a chromosome in map units