MD5019; Molecular genetics and

genomics

Coursework assignment: practical

research paper
J number:J101267
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Table of Contents

Abstract ............................................................................................................................... 3
Experiments 1 & 3 .............................................................................................................. 4
1. Introduction ............................................................................................................... 4

1.1. Polymerase Chain Reaction (PCR).................................................................... 4

1.2. Gel Electrophoresis............................................................................................ 4
1.3. Integrating Knowledge in Practical Settings ..................................................... 5

2. Methods..................................................................................................................... 5
3. Results and Discussion ............................................................................................. 7

3.1. Results ............................................................................................................... 7

3.1.1. Genotypic Frequency Calculation ..................................................................... 8
3.2. Discussion.......................................................................................................... 8

4. Conclusion ................................................................................................................ 9
Abstract ............................................................................................................................... 9
Experiment 2 ..................................................................................................................... 10
Introduction ....................................................................................................................... 10

DNA Polymorphisms and Their Detection ................................................................... 10

Polymerase Chain Reaction (PCR) and Gel Electrophoresis ....................................... 10
Practical Application and Analysis ............................................................................... 10

Methods............................................................................................................................. 11
Results and Discussion ..................................................................................................... 11
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Results ........................................................................................................................... 12
Discussion ..................................................................................................................... 12

Conclusion ........................................................................................................................ 13
References ......................................................................................................................... 13

Abstract

This lab report uses PCR and gel electrophoresis to study DNA
polymorphisms, particularly VNTRs and RFLPs. Our research focused on D1S80, a
diverse human DNA region. Diverse alleles indicate that the D1S80 locus is
employed in population genetics and genetic fingerprinting. Genotype frequencies
and allele sizes were measured precisely to mimic genetic variability patterns.
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Experiments 1 & 3
1. Introduction

The MD5019 extensively course on Molecular Genetics and Genomics covers DNA
polymorphisms, PCR, and gel electrophoresis via lectures, personal computer labs, and practical
components. DNA polymorphisms are population-specific sequence variations (Azizzadeh-
Roodpish et al., 2021). Variations may take various shapes. Genetic variations are important for
biodiversity and may be used in various studies. RFLPs and VNTRs, the most prevalent DNA
alterations, are its emphasis (NCBI. (2017). VNTRs are employed in genetic fingerprinting and
paternity testing. Restriction Fragment Length Polymorphisms (RFLPs) may alter where
restriction enzymes break DNA (Marshall et al., 2021). Their contributions are crucial to genetic
linkage studies that identify disease-related genes.

1.1.Polymerase Chain Reaction (PCR)

PCR is a cutting-edge method for replicating a short DNA sequence into many copies. In
genetic research, amplification is vital, particularly when the sample has restricted DNA. DNA
polymerase repeats the process of denaturation, annealing, and extension. The specificity of
polymerase chain reaction (PCR) is governed by primers (short DNA fragments) that identify the
targeted DNA area for amplification. This approach is essential for cloning, genotyping, gene
expression, and mutation detection in molecular biology (Mattes, 2019).

1.2.Gel Electrophoresis

The standard gel electrophoresis method separates DNA fragments by charge and size.
Students will learn agarose gel and polyacrylamide gel electrophoresis procedures after this course.
Agarose gel electrophoresis is necessary for RFLP pattern analysis and PCR product size
evaluation for bigger DNA fragments (Karunanathie et al., 2022). In VNTR studies,
polyacrylamide gel electrophoresis improves resolution and can distinguish small DNA fragments.
Electric fields move DNA fragments along the gel matrix. After reaching the gel matrix,
individuals are sized and evaluated to determine genetic diversity and structure.
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1.3.Integrating Knowledge in Practical Settings

For D1S80 polymorphism students employ DNA extraction, PCR amplification. Gel
electrophoresis is employed for the process of visualisation and analysis whole during practical
sessions. Linck & Battey, (2019) believe that hands-on learning may improve technical skills and
understanding of forensic science, molecular genetics, biotechnology, and genetic research of the
students. These lectures are among other PC courses. These sessions address genetic data
interpretation and provide software & computational tool use for data analysis. Mattes (2019)
stated that the course prepares students for future research by teaching them to apply molecular
genetics to real-world circumstances.

2. Methods

A table with example labels for each sample well helped to understand the results for the
Experiments 1 and 3. Due to strict allocation, DNA extraction, PCR amplification (using D1S80),
and gel electrophoresis (using agarose for Experiment 3 and polyacrylamide for Experiment 1)
were properly recorded in the documents appendix part (Zhu et al., 2020). Our extensive genomic
research allowed us to accurately anticipate migration distances and allele sizes.

Table 1 Migration Distances of Each Base Pair

Base Pairs Migration Distance (cm) Log of Base Pair Fragment Lengths
800 1.5 2.90308998699

750 1.55 2.87506126339

700 1.58 2.84509804001

650 1.6 2.81291335664

600 1.68 2.77815125038

550 1.7 2.74036268949

500 1.78 2.69897000434

450 1.8 2.65321251378

400 1.9 2.60205999133

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350 1.95 2.54406804435

300 2 2.47712125472

250 21 2.39794000867
200 2.25 2.30102999566
150 2.4 2.17609125906
100 2.65 2
50 3.15 1.69897000434

Table 2 Loading of Student DNA Samples

Lane Polymorphic Marker Sample Description

1 Base pair ladder DNA Ladder

2 D1S30 X

3 D1S80 X

4 D1S80 IM
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3. Results and Discussion

3.1.Results

Figure 1 Migration Distances

Figure 2 DS-180 Alleles

Focusing on the D1S80 polymorphism allowed us to clearly photograph our group gel for
Experiments 1 and 3. We calculated the migration distances of all marker bands and D1S80 alleles
in the sample using figure 1. Gel electrophoresis principles state that DNA band mobility on
polyacrylamide gel is inversely related to the logarithm of DNA molecular size (Schanfield et al.,
2023). We calculated ladder band migration distances to estimate our amplified samples' allele
band diameters using a standard curve. This was done using ladder bands. Linear regression
revealed the mathematical relationship between migratory distance and molecule size (Dwiningsih
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et al., 2022). Using the linear regression equation y = -0.243x + 3.4535 and a migration distance
of 2.5 cm, the computer could determine the band size. This magnitude is converted into D1S80
locus repeats in the final phase.

3.1.1. Genotypic Frequency Calculation

Each allele's repeat frequency was calculated using the standard curve. The Hardy-
Weinberg principle and data were used to compute the genotypic frequency of each group member
If no other factors contribute to evolution, genotype and allele frequencies in a population will stay
constant across generations. In homozygous condition (p2), Serrote et al., (2020) estimate that an
allele with a frequency of 0.1 (p) has a genotypic frequency of 0.01.

3.2.Discussion

Our sample has a wide range of D1S80 allele sizes, which matches human DNA variation.
We proved that the D1S80 marker is trustworthy for genetic diversity study by comparing our
findings to others on the same gel. Similar variability was reported in this investigation (Zhu et al.,
2020). Previous study has revealed that the D1S80 locus has a lot of variation, which supports our
results. Population genetics and genetic fingerprinting studies have revealed tandem repeat
variability's importance. D1S80 gene variation impacts several places. Variation enhancement may
aid paternity and forensics. Population genetics studies genetic diversity and evolution (Linck &
Battey, 2019).
Although promising, our results have limitations. According to Karunanathie et al. (2022),

our small sample size may make population genetics analysis problematic. Small allele size

variations may result from gel electrophoresis and PCR improvements. To study population

genetics, larger and more diverse samples may be used. Zhu et al. (2020) recommend

polymorphism markers to broaden the genetic profile. Finally, D1S80 polymorphism study

demonstrated the usefulness of genetic variation and molecular genetics. The findings implies

D1S80 has forensics and population genetics potential. Our study reveals this is possible. Although

restricted, our data supports genomics research in these sectors (Dwiningsih et al., 2022).
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4. Conclusion

This study revealed RFLP and VNTR DNA polymorphisms using PCR and gel

electrophoresis. Due to its variance, D1S80 may be utilised for population genetics and

fingerprinting. Gel electrophoresis matched gene size estimates. Hardy-Weinberg genotype

frequencies confirmed population genetic diversity.

Abstract

This lab report analyses two family pedigrees for genetic disease inheritance
patterns using Restriction Fragment Length Polymorphism (RFLP). Study found A2
and A3 consistently connected to the disorder, particularly in homozygous patients.
Genetic counsellors and researchers can understand these illnesses' genetic pathways
thanks to recessive inheritance. RFLP analysis is crucial in molecular genetics,
notably for hereditary disease diagnosis and prediction.
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Experiment 2
Introduction

Practical seminars, lectures, and computer-based instruction teach molecular genetics

basics. The programme covers DNA polymorphisms, identification, and impacts (Kadri, 2019).

DNA Polymorphisms and Their Detection

DNA polymorphisms are found utilising RFLPs and VNTRs to estimate population genetic

diversity. VNTRs appear in forensic and genetic linkage research. Due to high mutation and

polymorphism rates. Genomic DNA restriction enzymes modify structure, causing RFLPs. DNA

is distorted by enzymes. The enzymes can map hereditary illness genes (Gao et al., 2019).

Polymerase Chain Reaction (PCR) and Gel Electrophoresis

Presentations included gel electrophoresis and PCR. The lectures underlined the usefulness

of PCR for DNA sequencing and genetic marker research. Gel electrophoresis may discover

genome polymorphism patterns by size-selecting DNA fragments (Hashim & Al-Shuhaib, 2019).

Practical Application and Analysis

During practice, these methods were presented. By 2020, Trost and colleagues Students

analysed PCR-produced VNTR segments using gel electrophoresis. These technologies may be

used in genetic research and diagnostics since band pattern analysis may show genetic

heterogeneity. For their 2020 investigation, Serrote and colleagues did VNTR Experiment 1.

Genetic diversity markers and regional fingerprinting were recommended in this research. Future

RFLP research to find disease-related genes and map genetic information began with Experiment

2, which utilised concentrated RFLP analysis.

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According to Oldoni & Podini, (2019), the lectures taught students genetic analysis's basic
molecular methods. Lectures showed students how these methods are applied in forensic science,
medical genetics, and biodiversity research. Molecular genetics is a rapidly growing discipline that
requires these core principles and processes to progress knowledge and uncover new facts.

Methods

The main objective of this experiment is to shows how to monitor and identify samples in
each agarose gel well. This step is crucial because it links the observed DNA fragmenr to the
people from the two families under inquiry. A chart or graphic showing each sample's well is
essential. This graph shows gel electrophoresis findings (Hashim & Al-Shuhaib, 2019).

Results and Discussion

In the table below, family members with sick alleles and their fragments are shown.

Table 3 Family Member's Diseased Allele and Different Fragments Created in the Alleles

Generation Sex Diseased Fragments Fragments Allelic Genotype

Phenotype Produced in Produced in Form
Allele 1 (bp) Allele 2 (bp)
G1 M Yes 100, 500 600 A3, A4 Heterozygous
G2 M Yes 200, 400 100, 200, 300 A2, A2 Heterozygous
G3 F No 200, 400 200, 400 A2, A2 Homozygous
G4 F No 100, 500 100, 500 A3, A3 Homozygous
P1 M No 600 600 A4, A4 Homozygous
P2 M Yes 200, 400 100, 200, 300 A2, A1 Heterozygous
P3 F Yes 100, 500 600 A3, A4 Heterozygous
P4 F No 200, 400 200, 400 A2, A2 Homozygous
O1 M Yes 100, 500 100, 500 A3, A3 Homozygous
O2 M No 200, 400 200, 400 A2, A2 Homozygous
O3 M No 100, 500 100, 500 A3, A3 Homozygous
O4 F Yes 100, 500 600 A3, A4 Heterozygous
O5 F No 100, 500 100, 500 A3, A3 Homozygous
O6 F No 200, 400 200, 400 A2, A2 Homozygous
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O7 F Yes 200, 400 100, 200, 300 A2, A1 Heterozygous

Results

Experiment 2 RFLP analysis revealed two biological families of disease-related alleles.

The patterns generated by fragment sizes in alleles 1 and 2 for each person let us build reliable
family trees and analyze genetic linkage (Gao et al., 2019). Generation I men (G1 and G2) have
both homozygosity and heterozygosity for the sick phenotype. These men were A2, A4. Females
in good health with homozygous alleles (A2, A2) and (A3, A3) were classified as G3 and G4. P1
and P4 were healthy and homozygous (A4, A4) and (A2, A2) in the second generation, whereas
P2 and P3 exhibited illness signs and were heterozygous (A2, A1). People with at least one A4
allele in Generation III had no deleterious effects, comparable to the prior trend.

Discussion

Satapathy et al.'s 2019 study found that homozygous A2 or A3 allele carriers always had
the ill phenotype. This supports the pedigrees' claim that the illness was recessive. This
predisposition is stronger in Generations I and II because the undesired characteristic is linked to
two identical gene copies (Hashim & Al-Shuhaib, 2019). Our findings support the scientific
literature and show that Mendelian inheritance patterns cause genetic diseases. RFLP analysis
accurately predicts genetic vulnerability to certain illnesses. This research supports our
experimental results.
Some abnormalities were found, such as P2 and P3 people diagnosed with the illness while
having distinct genotypes. Pinta et al. (2023) suggest that the variations may be due to penetrance
and expressivity or a modifier gene. Since the genotypic distributions were substantially different
from a simple recessive model (p = 0.05), genetic complexity may be present. Statistical analysis
using chi-square tests confirmed this. Kadri, (2019) found that this families' inheritance pattern
has important implications for genetic counseling. This helps forecast and understand the
likelihood of a disease being passed on.
Experiment 2 efficiently used RFLP analysis to determine how genetic diseases are passed
down across families. Goswami et al. (2021) discovered ill phenotype-associated recessive
inheritance traits. These qualities were A2 and A3 alleles. Data anomalies indicate complexity and
need more examination. These results expand our knowledge of the genetics of the ailment and
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show molecular genetics' diagnostic and prognostic potential for hereditary conditions (Kadri,
2019).

Conclusion

The second experiment on two pedigreed families indicated a distinct genetic disease
inheritance pattern. RFLP testing showed that the sickness is recessive and that certain alleles are
associated with its prevalence. A2 and A3 consistently linked to the sick phenotype, especially in
individuals with two gene copies. These findings support genetic counselling and providing
relevant genetic information on visible features.

References

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Dwiningsih, Y., Rahmaningsih, M., & Alkahtani, J. (2020). Development of single nucleotide
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Linck, E., & Battey, C. J. (2019). Minor allele frequency thresholds strongly affect population
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