Techniques of Chromosomal Studies

Abstract

Chromosomal analysis has been an area of utmost significance for various cytogenetic and medical studies. The techniques to study normal and abnormal chromosomes which initiated as simple observations under the microscope with and without different types of dyes have now developed into more elaborate and invasive techniques. In the early years of cytogenetics, scientists had a difficult time distinguishing individual chromosomes, but over the years, they continued to refine the conditions for preserving and staining chromosomes to the reproducible standard that is now expected in clinical cytogenetics. In today’s procedures, metaphase chromosomes are treated with stains that generate distinctive banding patterns, and chromosome pairs are then arranged into a standardized format known as a karyotype. Over the past few decades, versatile methods based on fluorescence in situ hybridization (FISH) have transformed cytogenetics into a molecular science and provided cytogeneticists with powerful new tools. FISH procedures are now routinely employed in clinical cytogenetics. Although chromosomes may appear to be static structures when viewed under a microscope, cytogeneticists know that chromosomes are actually dynamic assemblies made up of a DNA-protein complex called chromatin. This chapter takes stock of all the prevalent techniques, highlighting the principals involved in each method. Karyotyping, genetic mapping, fluorescence in situ hybridization (FISH), multiplex FISH, spectral karyotyping, flow cytometry, and microarray have been described. New trends in cytogenetics to understand the molecular mechanism have been discussed under new generation sequencing.

Glossary

Acridine dyes

 A class of organic compounds that bind to DNA and intercalate into the double-stranded structure, producing local disruptions of base pairing. The disruptions result in additions or deletions in the next round of replication.

Allele

 One of the possible mutational states of a gene, distinguished from other alleles by phenotypic effects.

Aneuploidy

 A condition in which the number of chromosomes is not an exact multiple of the haploid set.

Atavism

 Reappearance of ancestral trait after several generations because of recessiveness of other masking effects.

Autism

 A group of complex disorders of brain development. These disorders are characterized, in varying degrees, by difficulties in social interaction, verbal and nonverbal communication, and repetitive behaviors.

Autopolyploidy

 Polyploid condition resulting from the replication of one diploid set of chromosomes.

Bacterial artificial chromosomes (BACs)

 Artificial chromosomes used for sequencing that are derived from bacterial fertility factors (F plasmids).

C-banding

 It is a technique in which the centromeric regions and other regions containing constitutive heterochromatin of the chromosomes are specifically stained.

Centimorgan

 A unit of distance between genes on chromosomes. One centimorgan represents a value of 1 % crossing over between two genes. Abbreviated as cM.

Centromere

 Specialized region of a chromosome to which sister chromatids remain attached after replication and the site to which spindle fibers attach during cell division. Location of the centromere determines the shape of the chromosome during the anaphase part of cell division. Also known as the primary constriction.

ChIP

 A method for purifying chromatin containing a protein of interest by immunoprecipitating the chromatin with an antibody directed against that protein or against an epitope tag attached to the protein.

Chromosomal aberration

 Any change resulting in the duplication, deletion, or rearrangement of chromosomal material.

Chromosome banding

 Technique for the differential staining of mitotic or meiotic chromosomes to produce a characteristic banding pattern or for the selective staining of certain chromosomal regions such as centromeres, the nucleolus organizer regions, and GC- or AT-rich regions. The pattern of banding is highly specific, and all the 23 human chromosomes can be identified. Not to be confused with the banding pattern in polytene chromosomes, which is produced by the alignment of chromomeres.

Chromosome map

 A diagram showing the location of genes on chromosomes.

Colchicine

 An alkaloid compound that inhibits spindle formation during cell division. Used in the preparation of karyotypes to collect a large population of cells inhibited at the metaphase stage of mitosis.

Comparative genomic hybridization (CGH)

 It is a molecular cytogenetic method for analyzing copy number variations (CNVs) relative to ploidy level in the DNA of a test sample compared to a reference sample, without the need for culturing cells.

CGH microarray

 Through the use of DNA microarray in conjunction with CGH techniques, the more specific form of array CGH (aCGH) has been developed, allowing for a locus-by-locus measure of CNV with increased resolution as low as 100 Kb.

Cleaved amplified polymorphic sequence (CAPS)

 It is a technique for the analysis of genetic markers. It is an extension to RFLP method, using PCR to more quickly analyze the results. The genetic differences between individuals can create or abolish restriction sites, and these differences can be detected in the resulting DNA fragment length after digestion. The digested PCR products will give readily distinguishable patterns of bands.

Congenital abnormalities

 Congenital abnormalities are caused by problems during the fetus’s development before birth.

Copy number variations (CNV)

 Large segments of DNA, ranging in size from thousands to millions of DNA bases, can vary in copy number. Genes that were thought to always occur in two copies per genome have now been found to be present in one, three, or more than three copies. In a few rare instances, the genes are missing altogether.

Crossing over 

Physical exchange between DNAs or chromosomes that occur during recombination.

Cytogenetics

 A branch of biology in which the techniques of both cytology and genetics are used to study heredity. This is a cytological approach to genetics mainly involving microscopic studies of chromosomes.

Cytological map

 A diagram showing the location of genes at particular chromosomal sites.

Developmental delay 

A developmental delay is any significant lag in a child’s physical, cognitive, behavioral, emotional, or social development, in comparison with norms.

Euchromatin

 Chromatin or chromosomal regions that are lightly stained and are relatively uncoiled during interphase of the cell cycle. Euchromatic regions contain most of the structural genes.

Fluorescence in situ hybridization (FISH)

 Fluorescence in situ hybridization – a technique in which a fluorescent dye is attached to a nucleotide probe that then binds to a specific site on a chromosome and makes itself visible by its fluorescence.

Flow cytometry

 It is a technology that is used to analyze the physical and chemical characteristics of particles in a fluid as it passes through at least one laser. Cell components are fluorescently labeled and then excited by the laser to emit light at varying wavelengths. It is a laser-based, biophysical technology employed in cell counting, cell sorting, biomarker detection, and protein engineering, by suspending cells in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and chemical characteristics of up to thousands of particles per second.

Fluorochromes or fluorescent dyes

 Fluorochromes absorb light. The absorption of light by a population of these molecules raises their energy level to a brief excited state. As they decay from this excited state, they emit fluorescent light.

Genetic markers

 Genes identifiable through phenotypic variants that can serve as points of reference in determining whether particular progeny are the result of recombination. These are alleles used as experimental probes to keep track of an individual, a tissue, a cell, a nucleus, a chromosome, or a gene.

Giemsa stain

 A complex of stains specific for the phosphate groups of DNA.

G-banding or Giemsa banding

 It is a technique used in cytogenetics to produce a visible karyotype by staining condensed chromosomes. It is useful for identifying genetic diseases through the photographic representation of the entire chromosome complement.

Haploid

 The chromosomal number in the gamete (n).

Haplotype

 A cluster of alleles on a single chromosome.

Heterochromatin

 Highly condensed chromosomal regions within which genes are usually transcriptionally inactive. It stains darkly even during interphase, often containing repetitive DNA with few genes.

Histones

 Small DNA-binding proteins with a preponderance of the basic, positively charged amino acids lysine and arginine. Histones are the fundamental protein components of nucleosomes. They function in the coiling of DNA in chromosomes and in the regulation of gene activity.

Idiogram

 A photograph or diagram of the chromosomes of a cell arranged in an orderly fashion.

Intercalators

 Class of chemical mutagenic compounds composed of flat, planar molecules that can sandwich themselves between successive base pairs and disrupt the machinery of replication, recombination, or repair.

Inversion

 Rotation of a segment of a chromosome by 180° so that the genes in this segment are present in the reverse order; characteristic inversion loops are produced during meiosis in the inversion heterozygotes.

Karyotype

 The visual description of a complete set of chromosomes in one cell of an organism; usually presented as a photomicrograph of an individual cell with the chromosomes arranged in a standard format showing the number, size, and shape of each chromosome type, used in low-resolution physical mapping to correlate gross chromosomal abnormalities the characteristics of specific traits of diseases.

Linkage

 The proximity of two or more markers on a chromosome; the closer together the markers are, the lower the probability that they will be separated by recombination, DNA repair, or replication processes, thereby increasing the probability that they will be inherited together. Genes are linked when the frequency of parental type progeny exceeds that of recombinant progeny.

Linkage groups

 Associations of loci on the same chromosome. In a species, there are as many linkage groups as there are homologous pairs of chromosomes.

Linkage number

 The number of times one strand of a helix coils about the other.

Loss of heterozygosity (LOH)

 It occurs when a somatic cell contains only one copy of an allele due to nondisjunction during mitosis, segregation during recombination, or deletion of a chromosome segment. LOH becomes critical when the remaining allele contains a point mutation that renders the gene inactive.

Macroarray

 Microtiter plate-based DNA array.

Marker

 A gene or mutation that serves as a signpost at a known location in the genome.

Maternal cell contamination (MCC) 

It is one of the risks associated with prenatal testing, which can occur when a fetal specimen comes into contact with maternal blood or tissue.

Microarray

 Small glass-slide DNA array. A chip containing many tiny spots of DNA or oligonucleotides used as a dot blot to measure expression of many genes at once.

Micro RNA (miRNA)

 A short (18–25 nucleotides) RNA produced naturally in cells that can control the expression of cellular genes by causing destruction of specific mRNAs or blocking their translation.

Microsatellite

 DNA element composed of 15–100 tandem repeats of one, two, or three base-pair sequences, such as CACACACA, dispersed throughout the eukaryotic genome. A given microsatellite is found in varying lengths scattered around a eukaryotic genome. The loci can be studied by polymerase chain reaction amplification.

Minisatellite

 DNA element composed of 10–40 bp tandem repeating units of identical sequences.

Microsatellite DNA

 Repeats of very short sequences of DNA.

Multiplex FISH

 M-FISH is a filter-based multicolor karyotyping technology, which provides the simplest way to label probes in a multiplex fashion as each probe fluorochrome is either completely absent or present. At least five distinguishable fluorochromes are needed for combinatorial labeling to uniquely identify all 24 chromosome types in the human genome using chromosome painting probe sets.

Nonhistone proteins

 The proteins remaining in chromatin after the histones are removed. They are mainly acidic proteins.

Phage artificial chromosome or P1-derived artificial chromosome (PAC) 

It is a form of chromosome derived through biological manipulation and it originates from a “phage” instead of a “plasmid.”

p arm of a chromosome

 The short arm of a chromosome. The “p” comes from the French “petit” meaning small. All human chromosomes have 2 arms – the p (short) arm and the q (long) arm – that are separated from each other only by a centromere.

Physical map

 Chromosomal map in which distances are in physical units of base pairs. These maps can be of microsatellite markers or of sequence-tagged sites.

Physical markers

 Cytologically visible abnormalities that make it possible to keep track of specific chromosomes parts.

Polyploidy level

 Polyploidy level describes cells with three or more sets of chromosomes.

q arm of a chromosome

 All human chromosomes have 2 arms – the p (short) arm and the q (long) arm – that are separated from each other only by a primary constriction. The symbol “q” was chosen for the long arm simply because it followed “p” in the alphabet.

Quantitative trait loci (QTLs)

 Loci that control the expression of continuous traits.

Q-banding

 In this technique, chromosomes are treated with quinacrine mustard solution, a fluorescent stain, to identify specific chromosomes and structural rearrangements. It is especially useful for distinguishing the Y chromosome and various polymorphisms involving satellites and centromeres of specific chromosomes.

R-banding or Giemsa reverse banding

 It is a technique in which the resulting chromosome pattern shows darkly stained R bands and pale G bands. This method is useful for analyzing deletions or translocations that involve the telomeres of chromosomes.

Random amplification of polymorphic DNA (RAPD)

 A protocol designed to detect single-base changes at polymorphic loci throughout a genome.

Recombination

 The production of gene combinations not found in the parents by the assortment of nonhomologous chromosomes and crossing over between homologous chromosomes during meiosis. For linked genes, the frequency of recombination can be used to estimate the genetic map distance; however, high frequencies (approaching 50 %) do not yield accurate estimates.

Restriction enzymes

 Proteins that recognize specific, short nucleotide sequences, and cut DNA at those sites. Bacteria contain over 400 such enzymes that recognize and cut over 100 different DNA sequences.

Restriction fragment length polymorphism (RFLP)

 Variation between individuals in DNA fragment size cut by specific restriction enzymes; polymorphic sequences that result in RFLPs are used as markers on both physical maps and genetic linkage maps.

Satellite DNAs

 Blocks of repetitive, simple noncoding sequences, usually around centromeres. These blocks have a different chromatin structure and different higher order packaging than other chromosomal regions.

Sequencing (high throughput or next generation)

 DNA sequencing that uses very rapid automated methods, such as pyrosequencing to produce relatively short reads of DNA in a massively parallel manner that yields a great deal of sequences in a short time.

Simple sequence repeats (SSR)

 A small segment of DNA, usually 2 to 5 bp in length that repeats itself a number of times.

SINE

 Sequences of DNA interspersed in eukaryotic chromosomes in many copies. Alu, a 300 base-pair sequence, is found about half a million times in human DNA.

Single-nucleotide polymorphisms (SNPs)

 Differences between individuals involving single base pairs that are located about every 1.000 bases along the human genome. SNPs are useful for mapping disease genes.

Tiling array 

Tiling arrays are a subtype of microarray chips. Like traditional microarrays, they function by hybridizing labeled DNA or RNA target molecules to probes fixed onto a solid surface. Tiling arrays differ from traditional microarrays in the nature of the probes.

Translocation

 A chromosomal rearrangement in which a segment of genetic material from one chromosome becomes heritably linked to another chromosome.

Variable-number-of-tandem-repeat (VNTR) loci

 Loci that are hypervariable because of tandem repeats. Presumably, variability is generated by unequal crossing over.

Whole genome sequencing (WGS)

 Also known as full genome sequencing, complete genome sequencing, or entire genome sequencing is a laboratory process that determines the complete DNA sequence of an organism’s genome at a single time.

Yeast artificial chromosome (YAC)

 A vector used to clone DNA fragments up to 400 kb; it is constructed from telomeric, centromeric, and replication origin sequences needed for replication in yeast cells.