500bp yields. Too much of primers increase the possibility

500bp

300bp

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50bp

150bp

1000bp

1500bp

2000bp

                                 

             

Figure 2a shows the band ladder measurement (in BP) which,
is used to determine the DNA fragment size in gel electrophoresis. Figure 2b
shows the band marker on lane 1 and 2 lanes of known DNA samples where both
lane 2 and lane 3 contain the 300-base pair DNA fragment.

Results

In this
practical, the band measurement was 300bp amplicon. The DNA fragments present in
both lanes are used to determine the number of repeat present according to
their length.

Multiple
bands were generated on both lanes due to non-specific application products. The
non-specific application product can be produced by primer dimer, too much
primers, DNA contamination or concentration of Mg2+ in the PCR however,
too little DNA template will have low yields. Too much of primers increase the possibility
of primers binding to nonspecific sites on the template. Moreover, high
concentrations of magnesium lead to nonspecific primer binding to unwanted
product formation. (ref primer dimer). Bands
below 50bp on agarose gel is primer dimer as they tend to self-anneal or anneal
to other primers in the reaction. Therefore, the concentration of primer need
to be the same as the template primer.

 

Discussion

Brief summary of results
and outcome of practical experiment.
Discuss the role of PCR
in forensic science and its use in criminal investigations.

PCR is crucial in forensic as only a small trace of evidence
is available for analysis. DNA sequence can be determined even if only small
amounts of biological sample obtained from the crime scene. Even forensic anthropologist
uses PCR to analyse ancient DNA that is 1000 years old

750bp

Figure
2a:
PCR ladder measurement.   Figure 2b: Direction of DNA migration

Result

 

 

 

 

 

 

5.    Electrophorese
the PCR products at 90V and after the run, remove the gel and visualise the PCR
fragment using UV light.

4.    20µl
of the PCR product was gently pipetted into the wells of the gel. Each well had
different PCR products. The first well contained PCR 50-2000bp which is a
marker in the gel loading buffer.

3.    40mls
of agarose gel was gently poured into the gel tray and the gel comb end was
inserted, which will help to form wells of the gel into which the sample will then
be pipetted. Making sure the gel was gently swirled and avoiding air bubbles.

2.    The
reaction components were mixed gently and was placed into icebox ready to be
centrifuge in a PCR thermocycler.

1.    5µl
calf thymus DNA, 5µl of primer 1, 5µl of primer 2 and 10µl of H2O
was added to the multi-mix solution in an eppendorf tube.

Material
& Method

 

Figure
1 shows the basic of PCR reaction when being used to amplify the targeted DNA
segment. The primers (shown in pink) are designed to bind to the end region of
the DNA fragment. This reaction is put through several temperature cycles;
denaturation at 95oc, annealing at 50-60oc and elongation
at 72oc. At each stage the amount of target DNA duplicates.

Figure 1:
The polymerase chain reaction (PCR)

 

 

 

To
synthesise DNA from RNA template, cDNA is produced by reverse transcription
ready to be amplified. The process for reverse transcription is similar to PCR
however the primer used is reverse transcriptase that degrades the RNA ready
for amplification. (reverse transcriptase) The amplified ? actin gene is then
visually detect through gel electrophoresis which is a technique used to separate biological
molecules with current. Agarose gel is used to separate DNA molecules based on their
sizes.

Elongation
is the final step of PCR. At 72oc Taq polymerase, is used to
synthesise the two new strands of DNA from the original strand as templates. At
this point the amount of the targeted DNA is duplicated, each of the new
molecules containing one old and one new strand of DNA. The cycle of denaturing
and synthesising new DNA is repeated, increasing to billion copies of the
original DNA segment (your genome). The cycling
process of PCR is automated and can be done in few hours using a thermocycler (Thermofisher).

To amplify
DNA found at a crime scene, the biological sample is heated so that the DNA
denatures into two pieces of single-stranded DNA. Denaturation occurs at 95oc
where the hydrogen bond of the double helix breaks, forming short sequences of two-single
stranded DNA. During the annealing stage, when the temperature is cooled to
50-60oc, the primer is attached to each end of the DNA template by
hydrogen bonding to be copied. The two DNA strand run opposite direction to
each other hence, there are one forward primer and a reverse primer. (search for book)

 

PCR
was invented in 1983 by Kary Mullis and has since become the most successful
technique for molecular genetics (forensic home
book). Kary Mullis discovered that, using two oligonucleotides allow DNA
to be synthesised from a single specific location in a genome. As from 1986,
PCR advanced significantly, centus scientists discovered that Taq polymerase, a
heat resistant enzyme and thermocycler could repeat the PCR cycling without
the need of adding DNA polymerase after each cycle, therefore human
intervention during the reaction process is not needed. Thermocycler was
invented in 1987, which is used to regulate the temperature of a reaction, such
as heating or cooling of samples (thermofisher
website).

PCR Background

 

Polymerase
Chain Reaction (PCR) is a technique used in DNA profiling and molecular
biology. PCR is used to amplify or make copies of a specific DNA region, as DNA
from biological samples might be limited at a crime scene. (KHAN ACADEMY) This technique therefore improves
the accuracy and speed of DNA detection.

Introduction