LEANNE M. SANTOS BS.

BIOLOGY 2-1 B

ACTIVITY 3
PRIMER DESIGN AND POLYMERASE CHAIN REACTION
(GENERAL BIOTECHNOLOGY LABORATORY)

OCTOBER 17, 2019

Questions to Answer:
1. Discuss the mechanism of thermal cycler.
A thermal cycle is an important device used in the process of Polymerase Chain Reaction
(PCR) to amplify target DNA sequences by altering the temperature of the reaction every few
minutes to allow DNA denaturing and synthesis. Modern thermal cyclers consist of thermal
blocks with holes in which tubes with PCR reaction mixtures are placed which raises and
lowers the temperature, and heated lid that is pressed against the lids of the inserted tubes to
prevent condensation of water from the reaction mixture. The steps that happen in this process
are the initial denaturation, denaturation by heat, annealing of primer to target sequence,
extension, and final extension which occurs at different temperatures.

2. Discuss the role/function and importance of each PCR reagents.

Polymerase Chain Reaction uses six chemical components that are each necessary for the
process of amplification. These six critical components are the template DNA, DNA
polymerase (Taq polymerase), forward and reverse primers, deoxynucleoside triphosphate
(dNTPs), PCR buffer (magnesium chloride), and sterilized water.
The first requirement - DNA template, is the particular DNA sequence that is going to be
copied. The optimal amount of PC amplification primarily depends on the composition and
complexity of the template DNA used, therefore it needs to be a high quality for the PCR to
have a high quality result. Second to this is the DNA polymerase which plays a critical role in
replicating the target DNA. Taq polymerase, in particular, incorporates nucleotides at a rate of
about 60 bases per second at 70 °C and amplify lengths of DNA to about 5 kb. Next component
are the forward and reverse primers. PCR primers are designed to bind to sequences that are at
the edge of the region of interest in the template DNA and ensure specific amplification of the
intended target. Another component for PCR is the dNTPs or deoxynucleoside triphosphate
that consist of four basic nucleotides which are the dATP, dCTP, dGTP, and dTTP which are
needed to provide the building blocks for DNA replication. Next is the PCR buffer - typically
consist of MgCl2, that is used to provide stable pH. Magnesium ions in buffer plays a vital role
in the PCR reaction by acting as a co-factor for Taq polymerase and thereby influencing
enzyme activity. Lastly, sterilized water or nuclease free water is used for PCR to lower the
risk of nuclease contamination in the PCR reaction mixture and to maintain the final volume
needed.

3. Differentiate dominant to co-dominant markers and functional to non-functional markers.

Codominant markers are markers for when both the alleles are expressed in an individual.
codominant markers can clearly discriminate between homozygotes and heterozygotes,
allowing the determination of genotypes and allele frequencies at loci. On the other hand,
dominant markers are scored as the presence or absence of fragments of a particular size,
therefore, heterozygosity is unable to be determined directly.
Functional markers are markers that are developed from the sequence polymorphisms
present within functional gene(s) which are associated with phenotypic trait variations. This
marker eliminates the problem associated with random DNA markers which helps in
improvements of crop breeding by selecting and pairing parental genotypes or eliminating
linkage drag in back-crossing, and also selecting traits that are difficult to measure using
phenotypic assays. While nonfunctional markers are developed from sequence from non-
coding region of DNA that has no effect on transcription rate. This molecular marker is said to
be ineffective in actual crop breeding.

4. What are molecular markers?

Molecular marker is a fragment of DNA that is associated with a certain location within
the genome. It is used to identify particular sequences of DNA from an unknown DNA pool to
reveal genetic variation. Molecular markers are also capable of identifying dominance and
codominance within a genome which helps to distinguish heterozygotes from homozygotes by
allowing amplification of particular sequence to be compared and analyzed. Molecular markers
basically help in identifying particular locations to a chromosome, thus allowing for physical
maps to be crated within long sequences of DNA. Examples of molecular markers include;
Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA
(RAPD), Amplified Fragment Length Polymorphism (AFLP), Variable Number Tandem
Repeat (VNTR), Oligonucleotide Polymorphism (OP), Single Nucleotide Polymorphism
(SNP), Allele Specific Associated Primers (ASAP), Inverse Sequence-tagged Repeats
(ISTR), and Inter-Retrotransposon Amplified Polymorphism (IRAP).
 METHODS

Steps for Polymerase Chain Reaction.

I. Preparation of PCR Cocktail:

1. Sterilized water (5 µl)
2. PCR buffer (2 µl)
3. Primer F (ITS1) (1 µl)
4. Primer R (ITS4) (1 µl)
5. dNTPs (2 µl)
6. Taq Polymerase (1 µl)
7. DNA sample (2 µl)

II. Process in Thermal Cycler:

1. Initiation (95°C for 3-5 min)
2. Denaturation (95°C for 30-60 sec)
3. Annealing (48-60°C for 30-60 sec)
4. Extension (72°C for 30-60 sec)
5. Final extension (72°C for 5-7 min)
III. Gel Electrophoresis:
1. Preparation of Agarose gel.
2. Casting the gel
3. Loading PCR product in gel
4. Gel viewing using documentation system.