Biosciences BB1704 Term 2: Molecular Analysis Practical Report

This practical report details a molecular analysis experiment conducted as part of the BB1704 course. The assignment focuses on the medical diagnostics of Single Nucleotide Polymorphisms (SNPs) using PCR, restriction enzyme digests, and agarose gel electrophoresis. The report is divided into two parts. Part 1 presents the results of PCR amplification of the β-globin gene from different genomic DNA samples, including a healthy individual, a β-thalassemia patient, and an individual undergoing diagnostic analysis. The results are visualized using agarose gel electrophoresis, and the size of the amplified fragments is estimated by comparing them with DNA molecular weight markers. Part 2 involves the restriction digestion of the PCR products with the NheI enzyme. The results are analyzed to determine the presence or absence of a specific restriction site, which indicates the presence of a mutation. The report includes detailed figure legends for each gel image, explaining the experimental setup, the observed results, and the conclusions drawn about the DNA samples analyzed. The report also discusses potential errors and the biological relevance of the findings, with the goal of diagnosing a specific genetic condition.

Part 2
Figure 1
Prepare Figure 1 showing the image of the gel obtained in the 1st part of BB1704 Term 2
practical and write the Figure Legend for this figure that includes the analysis of the results
of the Part 1 experiment.
In the figure, the lanes should be labelled with numbers (Lane 1, 2, 3…) at the top of the gel.
Each band of the HyperLadder (DNA molecular weight markers) on your gel should be
labelled with the corresponding sizes, similar to the HyperLadder I figure in the handout.
Label DNA bands using numbers (e.g., I, II, III) and arrows. If the experiment carried out by
your group did not work, you need to prepare two figures: Figure 1A showing the results of
your unsuccessful experiment with your explanation of what went wrong, and Figure 1B
showing the expected results and using the image of the gel provided by the supervisor (file:
Part 1 gel example). The proper figure legends should be written for both Figure 1A and
Figure 1B.
The figure legend should include: Figure Number; Figure Title (use bold or italic font to
distinguish the title from the rest of the figure legend); Brief explanation of what is shown in
the figure (which lane number corresponds to which sample). Include the conclusions for
Part 1 results in the Figure Legend. For this, make a visual comparison of migration of the
PCR products and the bands of HyperLadder to estimate the sizes of the DNA fragments
generated by PCR. Do they match the expected sizes? From which samples of genomic
DNA the PCR fragments were generated? Did the controls work? If the experiment did not
work for your group, and you have to prepare Figure 1A and Figure 1B, briefly reflect on the
most likely, in your opinion, errors.
Word limit – 150 words. If two figures are prepared, the word limit of each figure is 150
words.
Insert Figure 1 followed by the Figure Legend here:

1 2 3 4 5 6
I II III
Lane1: Template - genomic DNA from healthy individual
Lane2: Template - genomic DNA from β-thalassemia
patient
Lane3: Template - genomic DNA from the individual
undergoing diagnostic analysis
Lane4: Template - DNA from mouse
Lane5: Template - Distilled water used as template
(negative control)
PCR Amplified Products
Band I - β-globin gene from healthy
individual
Band II - β-globin gene from β-
thalassemia patient
Band III - β-globin gene from
individual undergoing diagnostic
analysis
1700bp 1700bp 1700bp

Figure 2
Prepare Figure 2 showing the image of the gel obtained in the 2nd part of BB1704 Term 2
practical and write the Figure Legend for this figure that includes the analysis of the results
of the Part 2 experiment.
In the figure, the lanes should be labelled with numbers (Lane 1, 2, 3…) at the top of the gel.
Each band of the HyperLadder (DNA molecular weight markers) on your gel should be
labelled with the corresponding sizes, similar to the HyperLadder I figure in the handout.
Label DNA bands using numbers (e.g., I, II, III) and arrows. If the pattern of bands in Lanes
1, 2, 3, 4 on your gel is different from the schematic presentation depicted in Fig.4B of the
Part 2 handout, the experiment carried out by your group did not work. In this case, you
need to prepare two figures: Figure 2A showing the results of your unsuccessful experiment
with your explanation of what went wrong, and Figure 2B showing the expected results and
using the image of the gel provided by the supervisor (file: Part 2 gel example). The proper
figure legends should be written for both Figure 2A and Figure 2B.
The figure legend should include: Figure Number; Figure Title (use bold or italic font to
distinguish the title from the rest of the figure legend); Brief explanation of what is shown in
the figure (which lane number corresponds to which sample). Include the brief explanations
for Part 2 results in the Figure Legend. Did you obtain a correctly-sized PCR fragments and
DNA fragments of the wild type and mutated DNA after restriction digestion with Nhe I?
Draw the conclusions about DNA X (DNA3). Comparing migration of the fragments after Nhe
I digestion of DNA No.1 (WT) and DNA No.2 (Mut) with DNA No. 3 (“X”) try to explain what is
represented by DNA “X” with respect to the biological relevance of analysed sample.
If the experiment did not work for your group, and you have to prepare Figure 2A and Figure
2B, briefly reflect on the most likely, in your opinion, errors.
Word limit – 150 words. If two figures are prepared, the word limit of each figure is 150
words.
Fig. 1 – Image of agarose gel electrophoresis results of the PCR amplified product of β-globin
gene from the five sample template. The target gene, β-globin, was successfully amplified in the
lanes 1 (genomic DNA template from healthy individual), 2 (genomic DNA template from β-
thalassemia patient) and 3 (genomic DNA template from individual undergoing diagnostic
analysis) and can be visualised as high intensity bands (I, II, III) at approximate size of 1700bp, as
deduced from marker bands of HyperLadderI in Lane6. This is in accordance with the known size
of the β-globin gene i.e. 1709bp. The amplification was not successful in Lane4 containing
genomic DNA template from mouse. Considering that all the PCR reagents, except the template
were identical in the five lanes, the probable reasons for no amplification may be degradation of
template DNA or incompatibility of primer with the template. The negative control with only
distilled water as template showed a clean lane with no amplification, hence confirming the
amplification of the target gene from the genomic DNA templates only.

Insert Figure 2 followed by the Figure Legend here:
Fig.2 – Image showing agarose gel electrophoresis results of NheI mediated restriction digestion of
wild type, mutant and test samples of PCR amplified β-globin gene. The restriction digestion of β-
globin amplified from wild type template showed a single band (1778bp), denoting absence of the
enzyme restriction site in this sample. Digestion of mutant sample yielded two fragments sized
1096bp and 602bp respectively (from calculations in part 3). A single nucleotide polymorphism in the
HBB gene courtesy of AG substitution results in appearance of NheI restriction site in the mutant
gene. The restriction enzyme introduced a nick at G^CTAGC. Finally, the digestion of the test sample
exhibited three fragments sized 1778bp, 1096bp and 676bp. The diagnostic deduction from afore
results is that the individual does not suffer from the disease but probably is a heterozygote for the
HBB allele. The control in each of the sets was the gene product without the NheI and for all the
three sets, a single fragment was identified at 1778bp, showing the presence of intact product in the
control.
1 2 3 4 5 6 7
II (1778bp)
I (1778bp)
IV (1096bp) II
V (676bp) II
III (1778bp)
IX (676bp)
II
VII (1778bp) II
VI (1778bp)
II
VIII (1096bp)
II
Set I – Wild Type (PCR product amplified from genomic DNA of healthy individual)
Lane 1- Negative Control (NC) - without NheI
Lane2- PCR product +NheI
Set II – Mutant (PCR product amplified from genomic DNA of β- thalassemia patient)
Lane3- Negative Control (NC) - without NheI
Lane4- PCR product +NheI
Set III –Test (PCR product amplified from genomic DNA of individual undergoing diagnostic
analysis)
Lane5- Negative Control (NC) - without NheI
Lane6- PCR product +NheI
Lane7- HyperladderI