BB1804 Biosciences: Practical Skills 3 - Molecular Analysis of DNA

This practical assignment focuses on the molecular analysis of DNA, specifically concerning the medical diagnostics of single nucleotide polymorphisms (SNPs) using PCR, restriction enzyme digests, and agarose gel electrophoresis. The assignment includes an abstract summarizing the coursework, which involves diagnostic testing for β-thalassemia via Nhe1-mediated restriction digestion of the β-globin gene. Results indicate a possible heterozygote for thalassemia based on observed DNA fragment patterns. The student also identifies and explains an incorrect example of a disease caused by a single nucleotide mutation, clarifying why the deletion of phenylalanine in CFTR is not a single nucleotide mutation but involves multiple nucleotide deletions. This analysis demonstrates practical skills in molecular biology and an understanding of genetic mutations and diagnostic techniques.

Part 1
Section 1: Abstract
Write an Abstract of 200 words (maximum) to explain your coursework. This should
include:
• Background: A simple opening sentence or two placing the work in context.
• Aims: One or two sentences giving the purpose of the work.
• Method(s): One or two sentences explaining what was done.
• Results: One or two sentences indicating the main findings.
• Conclusions: Evaluate the results of the Part 2 experiment – make the conclusion about
DNA X by comparing migration of the fragments after Nhe I digestion of DNA No.1 (WT) and
DNA No.2 (Mut) with DNA No. 3 (“X”).
Insert abstract here:
Single nucleotide polymorphisms (SNPs) account for approximately 0.1% variations in
human DNA and are determinant of diversity in population with regards to disease
susceptibility or response to drugs. SNPs in the HBB gene are commonly associated with
sickle-cell anemia and β-thalassemia.
The aim of the present study was to conduct a diagnostic test for β-thalassemia using
Polymerase Chain Reaction (PCR) and restriction digestion.
Diagnostic analysis was carried out for detection of β-thalassemia mutation by Nhe1
mediated restriction digestion of β-globin gene amplified from control, diseased and test
DNA.
The diagnostic analysis showed three bands upon NheI digestion (1778bp, 1096bp, 676bp),
while the wild type and mutant restriction samples exhibited one (1778bp) and two bands
(1096bp, 676bp), respectively; suggesting that this individual may be heterozygote for
thalassemia and not diseased.
The 1778bp fragment in the test group indicates its derivation from the non-mutant allele of
HBB, while the other two fragments seem to have been derived from mutant allele. Ideally
the sum of size of these two fragments should be 1778bp, however it is 6bp short. This
discrepancy may be due to rounding off of the values at each step of calculation to 2 decimal
places.
Section 2: Mutations causing the disease
Highlight the correct answer:

Which of three mutations is incorrect as an example of disease caused by a single
nucleotide mutation?
1) The deletion of phenylalanine in an amino acid position 508 (DeltaF508) in the cystic
fibrosis transmembrane conductance regulator protein encoded by the CFTR leading
to Cystic fibrosis disease.
2) The G to C mutation at amino acid 180 changes the codon TAC to TAG causing
termination of the polypeptide encoded by HEXA gene and leading to Tay-Sachs
disease.
3) The substitution of GAG to GTG in the 6th codon of the HBB gene, causing the
substitution of the amino acid glutamate to valine in β globin protein and leading to
Sickle Cell disease.
Provide a brief explanation of your answer here (maximum 50 words):
DeltaF508 in CFTR involves three nucleotide deletions - 3rd cytosine of Ile507 and 2 thymidines of
Phe508. This results in loss of Phe508 and a synonymous change in codon of Ile5071. Therefore
courtesy of 3 alterations instead of 1, this appears to be an incorrect example of a SNP disease.
Reference
1. Bartoszewski, R.A., Jablonsky, M., Bartoszewska, S., Stevenson, L., Dai, Q., Kappes, J., Collawn,
J.F. and Bebok, Z., 2010. A synonymous single nucleotide polymorphism in ΔF508 CFTR alters the
secondary structure of the mRNA and the expression of the mutant protein. Journal of Biological
Chemistry, 285(37), pp.28741-28748.