Medical Biotechnology (Biotech-630) Report on DNA Polymorphisms

This report provides a comprehensive overview of DNA polymorphisms, encompassing various types such as single nucleotide polymorphisms (SNPs), insertion/deletion polymorphisms (indels), polymorphic repetitive sequences (microsatellites), and structural and copy number variations (CNVs). It explores DNA-based molecular markers, including SNPs and microsatellites (STRs), and details techniques for their detection, such as restriction fragment length polymorphism (RFLP), polymerase chain reaction (PCR), genomic array technology, and sequencing. The report also examines the applications of these markers in mapping human diseases, predicting risk, quantitative trait loci mapping, pharmacogenetics, DNA fingerprinting, sex-chromosome STR testing, and DNA typing for engraftment monitoring. The content is supported by references to relevant scientific literature and provides a thorough understanding of the significance of DNA polymorphisms in various fields of biotechnology and medicine. This report is a valuable resource for students seeking information on the topic.

MEDICAL
BIOTECHNOLOGY
Course no:Biotech-630
Ramla Fatima
Seat no: 1746036

Date: 25-02-21
Contents
DNA Polymorphisms....................................................................................................................................2
Type of DNA polymorphisms.......................................................................................................................2
i. Single nucleotide polymorphisms....................................................................................................2
ii. Insertion/deletion polymorphisms..................................................................................................2
iii. Polymorphic repetitive sequences...................................................................................................2
iv. Structural and copy number variations...........................................................................................3
DNA-based molecular markers....................................................................................................................3
i. Single nucleotide polymorphisms....................................................................................................3
ii. Microsatellites (short tandem repeats)...........................................................................................3
Techniques for DNA-based molecular marker detection.............................................................................4
i. Restriction fragment length polymorphism.....................................................................................4
ii. Polymerase chain reaction...............................................................................................................4
iii. Genomic array technology...............................................................................................................4
iv. Sequencing......................................................................................................................................4
Application for DNA-based genetic markers................................................................................................5
i. Mapping human diseases and risk prediction..................................................................................5
ii. Quantitative trait loci mapping, candidate genes, and complex traits............................................5
iii. Pharmacogenetics...........................................................................................................................5
iv. DNA fingerprinting and human identification..................................................................................5
v. Sex-chromosome STR testing..........................................................................................................5
vi. DNA typing and engraftment monitoring........................................................................................5
REFERENCES................................................................................................................................................6
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DNA Polymorphisms
DNA polymorphisms are produced by changes in the nucleotide sequence or length. These result
from:
a. Variations in the fragment length pattern produced after digesting DNA with restriction
enzymes.
b. Variations in the size of a DNA fragment after PCR amplification.
c. Variations in the DNA sequence itself.
Since DNA from every tissue, from an individual show the same degree of polymorphism, they
become very useful identification tool in forensic applications. If an inheritable mutation is
observed in a population at high frequency, it is referred to as DNA polymorphism.
Type of DNA polymorphisms
i. Single nucleotide polymorphisms
Single nucleotide polymorphisms are the most common type of genetic variations in humans and
due to their abundance across the human genome; single nucleotide polymorphisms (SNPs) have
become important genetic markers for mapping human diseases, population genetics, and
evolutionary studies. SNPs are mostly formed when errors occur (substitution, insertion and
deletion) (Ismail & Essawi, 2012).
ii. Insertion/deletion polymorphisms
It is a type of DNA variation in which a specific nucleotide sequence of various lengths ranging
from one to several 100 base pairs is inserted or deleted. Indels are widely spread across the
genome. One base pair is considered as SNPs or repeat insertion/deletion as indels (Teama,
2018).
iii. Polymorphic repetitive sequences
DNA repeats can be classified as interspersed repeats or tandem repeats and can comprise over
two-thirds of the human genome.
Tandem repeats are organized in a head-to-tail orientation; based on the size of each repeat unit,
satellite repeats can be further divided into macrosatellites, minisatellites, and microsatellites.
Macrosatellites have sequence repeats longer than 100 bp and are the largest of the tandem DNA
repeats, located on one or multiple chromosomes. Minisatellites consists of stretches of DNA
and are characterized by moderate length patterns; 10–100 bp usually less than 50 bp.
Microsatellites are known as short tandem repeats (STRs) repeat units of less than 10 bp (Ismail
& Essawi, 2012).
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iv. Structural and copy number variations
Structural and copy number variations (CNVs) are another frequent source of genome
variability. The term CNVs therefore encompasses previously introduced terms such as large-
scale copy number variants (LCVs), copy number polymorphisms (CNPs), and intermediate-
sized variants (ISVs).
DNA-based molecular markers
Molecular markers are used to mark in genomes for various purposes such as mapping human
diseases, pharmacogenetics, and human identification.
i. Single nucleotide polymorphisms
Single nucleotide polymorphisms within a coding sequence cause genetic diseases including
sickle cell anemia. SNPs responsible for a disease can also occur in any genetic region that can
eventually affect the expression activity of genes, for example, in promoter regions.
Another important group of SNPs is the one that alters the primary structure of a protein
involved in drug metabolism; these SNPs are targets for pharmacogenetics studies.
SNV detection encompasses two broad areas:
a) Scanning DNA sequences for previously unknown polymorphisms and
b) Screening (genotyping) individuals for known polymorphisms. Scanning for new SNVs
can be further classified to two different types of approaches, the first one being the
global (or random approach) and the other one being the regional (targeted approach).
There are certain methods which have been developed for using SNVs randomly in the genome;
such as representation shotgun sequencing, primer-ligation-mediated PCR and degenerate
oligonucleotide–primed PCR (Teama, 2018).
ii. Microsatellites (short tandem repeats)
Microsatellites are short tandem repeats (STRs), repeat units, or motifs of less than 10 bp;
because of high variability, microsatellite loci are often used in forensics, population genetics,
and genetic genealogy. Variations in the STR length play a significant role in modulating gene
expression and STRs are likely to be general regulatory elements; regulatory STRs manifest
significant polymorphism because of their high intrinsic mutation rate (Krynetskiy, 2017).
Recently, polymorphic tandem repeated sequences and coy number variations have emerged as
important sources of genomic diversity that facilitate the study of genetic variations in health and
diseases.
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Techniques for DNA-based molecular marker detection
Different forms of DNA-based molecular markers can be tracked using a variety of techniques.
i. Restriction fragment length polymorphism
DNA digestion with restriction enzyme endonuclease cuts DNA at a specific sequence pattern
known as a restriction endonuclease recognition site. Thus, the alleles differ in length and can be
distinguished by gel electrophoresis, which can arise from a number of genetic events including
point mutation in restriction sites, mutation that creates a new restriction site, insertion, deletion,
and repeated sequences (Teama, 2018). Restriction fragment length polymorphism (RFLP) is a
type of polymorphism that results from variation in the DNA sequence recognized by restriction
enzymes (Restriction Fragment Length Polymorphism (RFLP),.)
ii. Polymerase chain reaction
In-vitro amplification of particular DNA sequences with the help of specifically chosen primers
and DNA polymerase enzyme is done. The amplified fragments are separated electrophonically
and detected by different staining methods. Real-time PCR useful modification of PCR can
detect polymorphisms by various methodologies using real-time PCR chemistries, for example,
TaqMan assay or molecular beacons (Teama, 2018).
iii. Genomic array technology
Genomic array technology is a type of hybridization analysis allowing simultaneous study of
large numbers of targets or samples. This technique uses Automated depositing systems
(arrayers) can place thousands of spots on glass substrate of the size of a microscope slide (chip)
with spotting representative sequences of each gene in triplicate, simultaneous screening of the
entire human genome on a single chip.
Microarray is also used to characterize genetic diversity and drug responses, to identify new drug
targets, and to assess the toxicological properties of chemicals and pharmaceuticals (Teama,
2018)
iv. Sequencing
Since technologies for rapid DNA sequencing have become available they are now widely used.
There is a great progression for the detection of single nucleotide variants (SNVs) by direct
sequencing.
Recent high-quality genome assemblies using long-read sequencing have revealed that each
human genome has approximately 20,000 structural variants, spanning 10 million base pairs,
more than twice the number of bases affected by SNVs (Pendleton et al., 2015).
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Application for DNA-based genetic markers
i. Mapping human diseases and risk prediction
The mapping of the human genome has made possible to develop a haplotype map in order to
better define human SNV variability. The haplotype map or HapMap will be a tool for the
detection of human genetic variation that can affect health and diseases (Sobrino et al., 2005).
ii. Quantitative trait loci mapping, candidate genes, and complex traits
The identification of genes affecting complex trait is a very difficult task. For many complex
traits, the observable variation is quantitative, and loci affecting such traits are generally termed
quantitative trait loci (QTL) (Xu et al., 2009).
iii. Pharmacogenetics
Individual response to a drug is governed by many factors such as genetics, age, sex,
environment, and disease. The influence of genetic factors on the response of a drug is a known
fact. Polymorphic STRs, together with SNPs and CNVs, can explain variability in response to
pharmacotherapy because of their prevalence in the human genome and their functional role as
regulators of gene expression and its applications (Teama, 2018).
iv. DNA fingerprinting and human identification
Establishing an individual’s identity is one of the uses of DNA sequence information that
highlights uniqueness of a particular sample, also known as genetic fingerprinting; DNA typing
and DNA profiling are molecular genetic methods that enable the identification of individuals
using hair, blood, semen, or other biological samples, based on unique patterns in their DNA.
Different DNA fingerprinting methods exist, using either restriction fragment length
polymorphism (RFLP) or PCR or both. Nowadays, in most forensic samples, the study of DNA
is usually performed by microsatellite analysis (Teama, 2018).
v. Sex-chromosome STR testing
Chromosome X specific STRs is used in the identification and the genomic studies of different
ethnic groups worldwide, because the small size of X-chromosome STR alleles; about 100–350
nucleotides, it is relatively easy to be amplified and detected with high sensitivity (Datta et al.,
2012).
vi. DNA typing and engraftment monitoring
DNA typing becomes the method of choice for engraftment monitoring, donor cells are
examined by following donor polymorphisms in the recipient blood and bone marrow (Teama,
2018).
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REFERENCES
Datta, P., Sood, S., Rastogi, P., Bhargava, K., Bhargava, D., & Yadav, M. (2012). DNA profiling
in forensic dentistry. Journal of Indian Academy of Forensic Medicine, 34, 156–159.
Ismail, S., & Essawi, M. (2012). Genetic polymorphism studies in humans. Middle East Journal
of Medical Genetics, 1, 57–63. https://doi.org/10.1097/01.MXE.0000415225.85003.47
Krynetskiy, E. (2017). Beyond SNPs and CNV: Pharmacogenomics of Polymorphic Tandem
Repeats. Journal of Pharmacogenomics & Pharmacoproteomics, 08(02).
https://doi.org/10.4172/2153-0645.1000170
Pendleton, M., Sebra, R., Pang, A. W. C., Ummat, A., Franzen, O., Rausch, T., Stütz, A. M.,
Stedman, W., Anantharaman, T., Hastie, A., Dai, H., Fritz, M. H.-Y., Cao, H., Cohain,
A., Deikus, G., Durrett, R. E., Blanchard, S. C., Altman, R., Chin, C.-S., … Bashir, A.
(2015). Assembly and diploid architecture of an individual human genome via single-
molecule technologies. Nature Methods, 12(8), 780–786.
https://doi.org/10.1038/nmeth.3454
Restriction Fragment Length Polymorphism (RFLP). (n.d.). Genome.Gov. Retrieved February
25, 2021, from https://www.genome.gov/genetics-glossary/Restriction-Fragment-Length-
Polymorphism
Sobrino, B., Brión, M., & Carracedo, A. (2005). SNPs in forensic genetics: A review on SNP
typing methodologies. Forensic Science International, 154(2–3), 181–194.
https://doi.org/10.1016/j.forsciint.2004.10.020
Teama, S. (2018). DNA Polymorphisms: DNA-Based Molecular Markers and Their Application
in Medicine. Genetic Diversity and Disease Susceptibility.
https://doi.org/10.5772/intechopen.79517
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Xu, J.-Y., Xu, G.-B., & Chen, S.-L. (2009). A new method for SNP discovery. BioTechniques,
46(3), 201–208. https://doi.org/10.2144/000113075
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