2 Neuroscience2.9 Genetics
2.9.4 Methods to identify genetic disorders and molecular genetic techniques
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Written by Resident Doctor Committee · Published
Chromosomes and the karyotype
A karyotype is a person's chromosome complement, defined by the number, size and structure of the chromosomes under the microscope. It is not the same as the genotype.
A chromosome counted at metaphase consists of two identical sister chromatids, made by replication in S phase and joined at the centromere.
Homologous chromosomes are the maternal and paternal copies of a pair.
Karyotyping
Karyotyping is usually done on cultured blood lymphocytes arrested at metaphase.
It is the definitive test for numerical disorders, such as Klinefelter syndrome and fetal aneuploidy, because it surveys every chromosome for number and structure, including balanced rearrangements.
G-banded karyotyping resolves only about 5 to 10 million bases, so it misses small deletions.
Cell-free DNA testing of maternal blood remains a screening test, not a diagnosis.
Fluorescence in situ hybridisation
Fluorescence in situ hybridisation (FISH) binds fluorescent probes to chosen sequences in metaphase chromosomes or interphase nuclei and shows the number and position of signals.
A locus-specific probe confirms a clinically suspected microdeletion, such as 22q11.2 or 7q11.23, when the expected signal is lost.
FISH answers only for the probed region.
Chromosomal microarray
Chromosomal microarray is the first-line genome-wide test for submicroscopic deletions and duplications, called copy number variants (CNVs), in unexplained developmental disorders.
It needs no suspected locus and no cell culture.
It is indicated in very early-onset schizophrenia, where the yield of pathogenic CNVs is high.
It misses balanced rearrangements, small sequence variants and low-level mosaicism, and it cannot size repeat expansions.
Sequencing
Test | What it reads | Limitation |
|---|---|---|
Gene panel | Curated disease-relevant genes | Blind to genes outside the panel |
Exome | Coding exons and splice boundaries | Misses regulatory and deep intronic sequence |
Whole genome sequencing discovers previously unknown variants anywhere in the genome when no candidate locus is known.
A person with intellectual disability and no diagnosis should be offered fragile X testing, chromosomal microarray and genome sequencing as routine, whatever their age or comorbidity.
Gel electrophoresis and PCR
In agarose gel electrophoresis, negatively charged DNA moves towards the anode and the gel slows longer fragments more, so DNA separates by size, not sequence.
A polymerase chain reaction (PCR) cycle has three steps, repeated to amplify the target exponentially.
Step | Temperature | What happens |
|---|---|---|
Denaturation | About 95°C | Heat separates the strands |
Annealing | About 50 to 65°C | Primers bind as the mixture cools |
Extension | About 72°C | Heat-stable polymerase extends the primers |
Replication in a vector and electrophoresis of the product are not part of the PCR cycle. Vector replication is cloning.
PCR across the repeat followed by electrophoretic sizing is the routine way to detect and size trinucleotide repeat expansions, but very large expansions need Southern blotting.
Cloning
A restriction endonuclease cuts DNA at a defined, often palindromic recognition site of 4 to 8 bases.
DNA ligase joins fragments with phosphodiester bonds.
A cloning vector, typically a bacterial plasmid, carries an isolated DNA insert into a host cell, where the insert is copied.
Blotting
Blot | Detects | Separation | Detected with |
|---|---|---|---|
Southern | DNA | Gel after restriction digestion | Labelled complementary probe |
Northern | RNA | Gel | Labelled complementary probe, showing gene expression |
Western | Protein | Polyacrylamide gel | Antibody, giving protein size and amount |
Southern blotting detects a known, long DNA sequence: restriction fragments are separated on a gel, transferred to a membrane and hybridised with a labelled probe.
Southern blotting shows fragment size with no copying needed, so it reveals expanded repeats and restriction fragment length polymorphisms.
Restriction fragment length polymorphisms arise when a variant changes a restriction site or the length between sites, and they can track a nearby disease locus through a family.
Gene silencing and gene editing
Knockdown lowers the gene product while the gene stays intact. A knockout or an edit changes the DNA.
Tool | How it works |
|---|---|
RNA interference | An endogenous pathway: Dicer cuts double-stranded or hairpin RNA into small interfering RNAs, which guide the RISC complex to cleave matching mRNA |
Morpholino oligonucleotide | Sits over the start codon and blocks translation, so protein falls while mRNA stays intact; used in zebrafish and Xenopus embryos |
Conditional (Cre-lox) knockout | Deletes DNA between two loxP sites only where Cre is made, confining loss to a cell type or period and avoiding embryonic death |
TALENs | Each repeat reads one base, chosen by its repeat-variable di-residue, and paired FokI domains cut both strands |
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. CRISPR-Cas is a prokaryotic immune defence against invading nucleic acids such as phages and plasmids.
The type II CRISPR system is the most widely used for genome editing. It has the single nuclease Cas9, a single guide RNA and an NGG PAM, and it makes a blunt double-strand cut.
The blood-brain barrier across the cerebral capillary endothelium is the main obstacle to delivering gene-editing components into neurons of the adult human brain.