3.7.3 Methods to identify genetic disorders

Methods to Identify Genetic Disorders

Genetic disorders are traditionally divided into three main categories: single-gene (or Mendelian) disorders, chromosomal disorders, and multifactorial disorders. Single-gene disorders, such as autosomal dominant, autosomal recessive, X-linked dominant, X-linked recessive, and Y-linked disorders, are caused by changes or mutations in the DNA sequence of a single gene. Chromosomal disorders, such as Down syndrome, are caused by errors in the number or structure of chromosomes. Multifactorial disorders, such as heart disease and diabetes, are caused by a combination of genetic and environmental factors.

The Greek words for colour (Chroma) and body (Soma) are where the word “chromosome” originates. Because chromosomes are cell structures or entities that are intensely stained by several vivid research dyes, scientists gave them this moniker (Meaburn & Misteli, 2007).

Diagnosis of genetic disorders can be done through various methods, including karyotype analysis, gene expression profiling, microarray analysis, and enzyme assays. Accurate identification of a genetic disorder is important for proper patient care, genetic counselling, and preventative measures.

There are several methods that can be used to identify genetic disorders:

Method for identifying genetic disorder:	Description:
Karyotype Analysis	This is a laboratory test that visually examines the chromosomes in a person’s cells to look for any abnormalities in their number or structure.
Gene Expression Profiling	This method uses high-throughput technologies to measure the levels of expression of thousands of genes simultaneously in order to identify changes that may be associated with a genetic disorder.
Microarray Analysis	This method is similar to gene expression profiling but uses a special type of DNA chip to analyze the expression of many genes at once.
Enzyme Assays	These tests measure the activity of enzymes, which are proteins that catalyze chemical reactions in the body. Decreased enzyme activity can indicate the presence of a genetic disorder.
DNA Sequencing	This method involves reading the entire DNA sequence of a person’s genes to look for mutations or changes that may cause a genetic disorder.
Biochemical Testing	This method measures the levels of specific substances in a person’s blood or other bodily fluids to identify genetic disorders caused by changes in the metabolism of these substances.
Clinical and Family History	A person’s clinical symptoms, as well as their family history, can also provide important information about the presence of a genetic disorder.
Prenatal Testing	Prenatal tests, such as chorionic villus sampling (CVS) or amniocentesis, can be used to diagnose genetic disorders in a developing fetus.

It’s important to note that some genetic disorders may be diagnosed using multiple methods for more accurate and comprehensive results.

Brief Explanations of Methodologies for Identifying Genes

There are several methodologies used to identify genes that are associated with genetic disorders. Some of these include:

Method for identifying genes:	Description:
Linkage Analysis	This method looks at the inheritance patterns of a genetic disorder within families to identify the location of the gene responsible for the disorder.
Candidate Gene Approaches	This method is based on prior knowledge of the biology of the disorder and involves testing specific genes that are thought to be involved in the disease based on their function, location, and other factors.
Genome-Wide Association Studies (GWAS)	This method involves comparing the DNA of many individuals with and without a particular disorder to identify common genetic variations that are associated with the disease.
Exome Sequencing	This method involves sequencing only the protein-coding regions of a person’s genes, which make up only a small fraction of the genome but contain most of the genetic information responsible for causing disease.
Whole Genome Sequencing	This method involves sequencing the entire genome of a person, including both the protein-coding regions and the non-coding regions, to identify any genetic changes that may be associated with a disorder.
RNA Sequencing	This method measures the levels of RNA, which is the product of gene expression, in a person’s cells to identify changes that may be associated with a disorder.

Each of these methodologies has its own strengths and limitations, and the choice of which one to use depends on the specific genetic disorder being studied and the research questions being asked.

Computational tools:

GRAIL, GENSCAN, and HMM are computational tools that are used in bioinformatics to identify genes in DNA sequences.

1. GRAIL (Gene Recognition and Analysis Internet Linked) is a gene-finding program that uses a combination of algorithms to predict the location of genes in DNA sequences. It takes into account both the coding potential of a DNA sequence as well as other factors, such as the presence of splice sites and promoter regions.
2. GENSCAN (Gene Structure Analysis) is a gene-finding program that uses a statistical model to predict the locations of genes in DNA sequences. It takes into account the patterns of codons and the frequency of start and stop codons in the DNA sequence to identify likely gene regions.
3. HMM (Hidden Markov Model) is a statistical model that is commonly used in gene prediction. It is based on the idea that genes have certain characteristic patterns, such as codon usage, that can be modelled mathematically. HMMs can be trained on known gene sequences to identify new genes in uncharacterized DNA sequences (Hänzelmann, 2013).

These tools have been widely used in the field of bioinformatics and have helped to identify many genes and genomic regions associated with various genetic disorders. However, they are only as good as the algorithms and models they are based on, and their predictions must be validated experimentally to confirm their accuracy.

Techniques in Molecular Genetics

Molecular genetics is a field of biology that deals with the study of genes and their functions at the molecular level. There are several techniques that are commonly used in molecular genetics to study genes and the changes that occur in them:

1. Polymerase Chain Reaction (PCR): This is a powerful and versatile technique used to make many copies of a specific DNA sequence. It is used to amplify small amounts of DNA for further analysis, such as sequencing or genetic testing.
2. DNA Sequencing: This is the process of determining the exact order of nucleotides in a DNA molecule. It is used to identify mutations or changes in DNA that may be associated with genetic disorders.
3. Restriction Fragment Length Polymorphism: This is a technique used to detect differences in DNA sequences between individuals. It involves cutting DNA with restriction enzymes, which produce fragments of different lengths. These fragments can then be separated by size and compared to identify differences in the DNA sequence.
4. Southern Blotting: This is a technique used to detect specific DNA sequences in a mixture of DNA. It involves separating DNA fragments by size and transferring them onto a piece of nitrocellulose or nylon membrane. The DNA is then probed with a labelled DNA sequence to identify the specific fragment of interest.
5. Gel Electrophoresis: This is a technique used to separate DNA, RNA, or protein molecules based on their size. It involves applying an electrical current to a mixture of these molecules, which causes them to migrate through a gel matrix. The resulting pattern of separated molecules can be used to identify specific molecules or compare differences between samples.
6. Cloning: This is the process of creating identical copies of a piece of DNA. It is used to study the function of specific genes and to produce large amounts of DNA for further analysis.
7. Gene Expression Analysis: This is the process of measuring the levels of gene expression, or the amount of a specific protein produced by a gene, in a sample of cells. It is used to study the regulation of gene expression and to identify changes in gene expression that may be associated with genetic disorders.

These techniques play a crucial role in the study of molecular genetics and have helped to advance our understanding of the genetic basis of many diseases.

Restriction enzymes:

Natural endonucleases known as restriction enzymes are utilised in molecular biology to cut DNA sequences at particular locations. Through ligation, DNA fragments with similar ends can be linked. The molecule is referred to as recombinant if the ligation results in a sequence that cannot be found in nature.

Naturally occurring restriction endonucleases are categorized into five groups (Types I, II, III, IV, and V):

Type V restriction enzymes (e.g., the cas9-gRNA complex from CRISPRs) utilize guide RNAs to target specific non-palindromic sequences found on invading organisms. They can cut DNA of variable length, provided that a suitable guide RNA is provided. The flexibility and ease of use of these enzymes make them promising for future genetic engineering applications.

Blotting methods:

In a mixture of DNA, Southern blotting can be used to find any sequence that matches a probe (such as total genomic DNA). Whereas RNA can be identified with Northern blotting. Finally, Western blotting can identify protein fragments.

Restriction Fragment Length Polymorphism (RFLP):

Restriction Fragment Length Polymorphism (RFLP) is a technique used in molecular genetics to detect differences in DNA sequences between individuals. It is based on the observation that variations in DNA sequences can result in differences in the length of fragments produced when the DNA is cut with restriction enzymes.

Here’s how RFLP works:

1. DNA is extracted from a sample and cut into fragments using specific restriction enzymes.
2. The fragments are separated by size using gel electrophoresis.
3. The separated fragments are then transferred onto a nitrocellulose or nylon membrane.
4. A probe, which is a labelled piece of DNA complementary to a specific sequence, is then used to identify the specific fragment of interest on the membrane.
5. If the probe hybridizes with the fragment, it will be visible on the X-ray film as a band.
6. The size of the fragment and the pattern of bands can then be compared between different samples to identify differences in the DNA sequence.

RFLP has been widely used in the study of human genetics, particularly in the fields of forensic science, medical genetics, and population genetics. It has also been used to study the evolution and diversity of different species.

However, RFLP has been largely replaced by more advanced techniques such as polymerase chain reaction (PCR) and DNA sequencing, which provide more accurate and detailed information about DNA sequences (NBCI, 2023).

Genetic recombination:

Recombination is the process by which fragments of DNA are broken and recombined to create new allele combinations. This recombination process generates genetic variety at the gene level, reflecting changes in DNA sequences between organisms.

References:

(1) Hänzelmann, S., Castelo, R. and Guinney, J. (2013). GSVA: gene set variation analysis for microarray and RNA-Seq data. BMC Bioinformatics, 14(1), p.7. doi:https://doi.org/10.1186/1471-2105-14-7.

‌(2) Meaburn, K.J. and Misteli, T. (2007). Chromosome territories. Nature, 445(7126), pp.379–381. doi:https://doi.org/10.1038/445379a.

‌(3) NCBI, 2023. www.ncbi.nlm.nih.gov. (n.d.). Restriction Fragment Length Polymorphism (RFLP). [online] Available at: https://www.ncbi.nlm.nih.gov/probe/docs/techrflp/#:~:text=is%20a%20difference%20in%20homologous.