- 3.1 Introduction
- 3.2 Cell Culture Medium: Peripheral Blood Lymphocytes for Chromosome Studies in Humans
- 3.3 Chromosome Banding and the Human Karyotype
- 3.4 Cytogenetic Approaches to Map Genes
- 3.5 Fluorescence in Situ Hybridization (FISH)
- 3.6 Advances in Molecular Cytogenetic Analysis
- 3.6.1 Whole Chromosome Painting and M-FISH
- 3.6.2 Spectral Karyotyping (SKY)
- 3.6.3 Comparative Genomic Hybridization (CGH)
- 3.6.4 Array CGH (aCGH)
- 3.7 Flow Karyotyping
3.1 INTRODUCTION
The branch of genetics which deals with the study of the structure and function of the cell, especially the chromosomes is known as Cytogenetics. Cytogenetic analysis can be carried out virtually for any cell in the body. However analysis of certain cells such as lymphocytes yield the best quality of chromosomes for study. Generally to undertand the complete chromosomal complement, Karyotyping is done. A Karyotype is defined as the identification of chromosomes based on their size, centromere location and banding pattern. When the chromosomal image is organized based on the same criteria then we get an Ideogram. The normal
human karyotype is written as 46, XX for females and 46, XY for Males. The nomenclature and correct method of denoting the karyotype, normal chromosomes and aberrations (which have been described in Unit 2) are done in accordance to the International Society for Chromosome Nomenclature (ISCN) guidelines.
3.2 CELL CULTURE MEDIUM: PERIPHERAL BLOOD LYMPHOCYTES FOR CHROMOSOME STUDIES IN HUMANS
As the culture is essentially set up outside a living system in a laboratory and under artificial conditions, it is critical that the cells be made to ‘feel at home’. Hence macro-environmental conditions such as pH, Temperature, O2/ CO2 concentrations, humidity and sterility have to be carefully maintained while setting up the cell cultures. In addition to this, other micro environmental conditions like nutrients, growth factors, signal molecules etc., should also be carefully regulated.
Peripheral blood forms an ideal source for studying Human Chromosomes. This is because it contains lymphocytes and is easily collected without much discomfort to the subject. These types of cultures are of the primary type as whole blood is used, the culture is finite i.e the cells are only viable for a maximum period of 72 hours after it is initiated. These cells have large nuclei and yield high quality Metaphases for analysis. Also genetic damages if present can be easily observed on analysis. However the drawback of using these cells is that they are mature cells. In in vitro cultures often a mitogen be used. The function of mitogen is to stimulate division of the lymphocytes that are under culture. One commonly and sucessfully used mitogen is Phytohemaglutinin (PHA) an extract from the plant Phaseolus vulgaris. During culture in order to arrest the cells at metaphase certain mitotic spindle inhibitors like Colchicine or Methotrexate are added at two to four hours before the termination of the culture.
RPMI 1640 has traditionally been used for the serum-free expansion of human lymphoid cells. (RPMI- Roswell Park Memorial Institute). It has traditionally been used for growth of Human lymphoid cells. This medium contains a great deal of phosphate and is formulated for use in a 5% carbon dioxide atmosphere. Generally a bicarbonate buffer is required. However several modifications are available. pH indicator is Phenol Red.
3.3 CHROMOSOME BANDING AND THE HUMAN KARYOTYPE
Maximo Drets and Margery Shaw established methods to stain metaphase chromosomes using a dye called Giemsa, which produces a signature banding pattern, called G-bands, for each of the 24 different human chromosomes. Gbanding patterns can be used to detect chromosomal translocations, deletions, and insertions, and localising the genes to specific regions of the chromosomes.
Chromosomal Banding Patterns: Chromosomes are composed of chromatin. Chromatin is nothing but the DNA Polynucleotide encased within several different proteins including Histones. Chromatin itself is of different types; the more activeregions are known as Euchromatin where as the less functional and more structural regions are known as heterochromatin. On staining ‘Banding’ pattern that is specific to each chromosome is observed that helps in identifying the chromosmes and arranging them in the form of karyotypes. The differential pattern of staining of chromosomes occurs with the additional treatment of the cultures with proteolytic enzymes such a Trypsin, Alkali or even Heat.
Banding techniques are often referred to by a single alphabet (such as G, R, C, Q, NOR) that denotes the type of banding and more precisely by three alphabets that denote: 1) The type of banding observed, 2) The treatment being used and 3) by the stain being used.
For example: G Banding is also known as GTG banding indicating
- G for G Banding
- T for Trypsin when used in culture
- G for Geimsa stain when used in culture
a) G-banding: This technique does not involve a fluorochrome-based pretreatment. During mitosis, the 23 pairs of human chromosomes condense and are visible under a light microscope. A karyotype analysis usually involves blocking cells in mitosis and staining the condensed chromosomes with Giemsa dye at metaphase stage. The dye stains regions of chromosomes that are AT rich (i.e. rich in the DNA base pairs Adenine and Thymine) producing a dark band. The G-light bands are thought to be relatively GC-rich (rich in the DNA base pairs guanine and cytosine). Furthermore, the light bands represent the regions which are relatively open and which contain most of the genes, including house keeping genes (genes active in every cell type). On the other hand, the G-dark bands represent regions which are relatively compact and contain few genes. The genes in the dark regions are mainly tissue-specific (Fig. 3.1).
b) R-banding: This method involves a moderate use of alkaline with or without heating to obtain a banding pattern that is reverse to G Banding. Here the AT rich region with more heterochromatin is lightly stained and the more active GC regions form the dark bands. In this method centromeric regions do not take up staining.
c) C-banding: In this method, method of staining only the constitutive heterochromatin regions of chromosomes is stained. That is only the centromeres and the satellite region on chromosomes gets darkly stained. Hot alkali is used to accomplish this effect; treatment with same would bring about depurination of DNA at regions that are vulnerable. As constitutive heterochromatin represents the most resistant region on the chromosome to such treatment, it remains intact and is stained.
d) Q-banding: This banding pattern is obtained by treating with a fluorochrome or the fluorescent dye quinacrin. They can be identified by a yellow fluorescence of different intensity. Most parts of the stained DNA are heterochromatin. Quinacrin binds those regions which are rich in AT and GC, but fluorescences only A-T-quinacrine regions. A-T regions are seen more in heterochromatin than in euchromatin. Therefore, by this banding method heterochromatin regions are labeled preferentially. The characters of the banding regions and the specificity of the fluorochrome are not exclusively dependent on their affinity to regions rich in A- T, but it depends on the distribution of A- T and its association with other molecules such as histone proteins.
e) NOR Banding: Human chromosomes belonging to the D (13, 14 and 15) as well as the G (21 and 22) group of chromosomes contain secondary constrictions called satellites. These regions are in fact the Nucleolar Organizing Regions, which come together to form the Nucleolus. This is the site for rRNA synthesis- which is the most abundant type of RNA found in mammalian cells. Here in addition to alkali treatment, Sivler Nitrate staining is used which gets preferentially deposited in these regions, making them darkly stained.
3.4 CYTOGENETIC APPROACHES TO MAP GENES
The very nature of cytogenetic analysis lends to identifying the location of gene i.e. Gene Mapping. This is because of the fact that most disorders are caused by mutations in genes. If the mutation involves a chromosomal aberration, then it follows that by identifying the defect we can trace the location of the gene on to a specific chromosome. Cytogenetic approaches in addition to providing associations, between chromosomal abnormalities and disease, have also allowed researchers to map genes to particular chromosomes. However in conventional chromosomal analysis some of the following limitations are:
- Low resolution limit: Unable to identify subtle chromosomal aberrations such as microdeletions and cryptic translocations, both of which involve very small chromosomal segments but are the genetic cause for disease.
- Inability to identify copy number variants that arise due to gene duplications or deletions.
- Delay in culturing cells to understand presence or absence of a gene.
3.5 FLUORESCENCE IN SITU HYBRIDIZATION (FISH)
Overview
It essentially involves the hybridization of a genetic ‘probe’sequence to its complimentary region in the human genome.In order to accomplish this, the target chromosomes are first denatured. The probe of course is composed of DNA/ RNA or cDNA from the gene of interest. The probe is essentially a stretch of labeled oligonucleotide that is used to identify the location of a gene on a chromosome. The principle of this method lies in choosing probes that have a greater specificity.
In early work, probes were labeled with radioactive isotopes and target sequences were identified by autoradiography. This method of labeling and detection limits both the sensitivity of the technique and its resolution. In particular, the original protocol only allowed the detection of tandemly repeated sequences such as the ribosomal genes and satellite DNA. By 1981, however, investigators had optimized the insitu protocol for use in mapping single copy mammalian sequences, and in 1984 an improved method was developed for better resolution of chromosome banding patterns.
Nevertheless, the technique is still not ideal because with single-copy radioactive probes, localization of genes can not be determined within the chromosomes of a single cell; instead, it is necessary to perform a statistical analysis of silver grain distributions in 50-100 sets of metaphase chromosomes.
Two critical changes in the protocol now allow the detection of single-copy sequences and their high-resolution mapping through the direct observation of single chromosomes. The first change made is in the nature of the label with the substitution of fluorescent tags for radioactive ones and dramatic improvement of the physical resolution of the hybridization site. The modified in situ protocol that utilizes fluorescent tags is referred to as FISH (fluorescent in situ hybridization).
The second change was in the nature of the hybridization cocktail. With the inclusion of a large excess of unlabeled total genomic DNA, it is possible to block dispersed repetitive sequences — present in essentially every genomic region larger than a few kilobases in length — from hybridization to their targets throughout the genome.
FISH (fluorescence in situ hybridization) is a cytogenetic technique developed by biomedical researchers in the early 1980s that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence complementarity. FISH is often used for finding specific features in DNA for use in genetic counselling, medicine, and species identification. FISH can also be used to detect and localize specific mRNAs within tissue samples. In this context, it can also help define the spatialtemporal patterns of gene expression within cells and tissues.
The FISH method involves four steps: fixation, hybridization, washing, and detection
Types of Probes
A probe is a stretch of DNA sequence that hybridizes with DNA/RNA based on the complementary base pairing property. When probes developed from known sequence of a gene hybridize with test DNA sample, it indicates the presence of the complementary sequence or the gene in the sample tested. The different types of probes are:
a) Locus specific probes: These probes bind to a particular region of a chromosome. This type of probe is useful when scientists have isolated a small portion of a gene and want to determine on which chromosome the gene is located.
b) Repeat binding probes: These probes bind to part of the human genome that contains certain types of repeats. Some such elements include Centromeric, Telomeric and Alu repeat (a type of transposon) probes. These probes are used to detect the presence of the repeats and detect centromere related aberrations.
c) Whole chromosome probes: They actually are collections of smaller probes, each of which binds to a different sequence along the length of a given chromosome. Using multiple probes labeled with a mixture of different fluorescent dyes, scientists are able to label each chromosome in its own unique color. The resulting is a full-color map of the chromosome. Whole chromosome probes are particularly useful for examining chromosomal abnormalities by screening the whole genome-for example, when a piece of one chromosome is attached to the end of another chromosome.
d) Arm specific probes: These probes hybridize to unique sequences either p or q arms of all chromosomes (except p-arm of acrocentric chromosome). Their use in molecular cytogenetic examination of patients include analysis of chromosome patterns involved in translocations, to study mutagenesis in human chromosomes, analysis of complex chromosomal rearrangments in
e) Band specific probes: Band specific probes are capable of detecting small chromosomal segments those involved in subtle translocations with break points. These particular probes increase the resolution typically obtained with whole chromosome probes when identifying chromosomal abnormalities.
The NOR probe is specific for p-arm of acrocentric chromosome, where as the Alu probe will detect Alu repeat sequences found in primate chromosomes.
3.6 ADVANCES IN MOLECULAR CYTOGENETIC ANALYSIS
After the development of FISH several advances in FISH based methodology were developed, some of these are described below.
3.6.1 Whole Chromosome Painting and M-FISH
In some cases it is advantageous to label larger segments of chromosome such as part of a arm, one arm or the entire chromosome itself. All these can then be used to track chromosomal aberrations including translocations (as illustrated in Fig. 3.6). However it is not possible to span the entire chromosome using a single probe. Hence chromosome paint probes are used. These are probes that contain overlapping ends such that they will hybridize at different points along the desired target chromosome giving a fluorescent label to the entire region. By using several combinations of probes each chromosome in the human genome can be given a separate color. Such a combinatorial approach is known as M-FISH or Multicolor FISH. Spectral Karyotyping (SKY) and Comparative Genomic Hybridization (CGH) are types of M-FISH.
The figure shows a translocation of SRY material to the q-arm of the del(X). The X chromosome is painted in green and the Y chromosome in red/orange. Normal cross hybridization of the Y painting probe is seen in proximal Xq of both the normal X chromosome and the del(X), whereas normal cross hybridization to Xp is only seen in the normal X chromosome as these sequences are missing from del(X). The del(X) also shows a signal at distal Xq, corresponding to translocated Y sequences. Inset (A): SRY material (red/orange) is located at distal Xq while X centromere is in green. Inset (B): The G- banding of del (X) and normal X.
Advantages
- 1) Easy to detect structural and numerical aberrations.
- 2) Each chromosome can be given a different label allowing screening of the entire genome.
Disadvantage
- 1) Costly to label the entire genome using probe combinations.
- 2) Cannot detect paracentric inversions.
3.6.2 Spectral Karyotyping (SKY)
Spectral karyotyping is a molecular cytogenetic technique used to simultaneously visualize all the pairs of chromosomes in an organism in different colors. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. Because there are a limited number of spectrally-distinct fluorophores, a combinatorial labeling method is used to generate many different colors. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope; this device is used to distinguish minor changes in the fluorescent signal that cannot be observed by the human eye.
Then the image processing software assigns pseudocolors to each spectrally different combinations, allowing the visualization of the individually colored chromosomes. Hence this method can be used as a screening method for analyzing the entire chromosomal compliment at once.
Advantages
- 1) A sensitive method, can detect complex translocation involving two or more chromosomes.
- 2) Provides a critical screening method that can analyze all the chromosomes at once.
Disadvantages
- 1) Cannot detect inversions especially paracentric inversions.
- 2) Due to the nature of the probes they are very expensive.
3.6.3 Comparative Genomic Hybridization (CGH)
This method is based on ratiometric analysis. This method uses the following approach:
- 1) DNA is extracted from the sample that needs to be tested. It is labeled using a fluorescent dye (such as TRITC- Tetramethyl Rhodamine Isothiocyanate) by the Nick Translation method.
- 2) A suitable control is also taken; this too is labeled but with a distinctly different colored fluorescent dye (for. Ex. FITC- Fluoresce in Isothiocyanate which gives a green color).
- 3) Both these labeled DNAs will then be hybridized on to a slide containing Normal Human Metaphase Chromosomes.
- 4) Once the hybridization is completed the analysis is done.
Here the analysis is done by comparing the test or sample DNA as compared to the control DNA.
Three outcomes are possible while applying this procedure. They are:
- 1) No loss or gain of genetic material in a particular region. This results in a balanced color being formed and balance between both the dyes (in this case it would appear orange= Red labeled DNA = Green labeled DNA).
- 2) Regions which have been deleted in the test sample will be showing a greater signal of the normal DNA. In this case it would result in a red signal. (Green label DNA< Red Labeled DNA).
- 3) Region where there has been a gain of genetic material in the sample DNA as compared to the normal DNA. In this case it would show an excess of the sample’s fluorescent color (Green label DNA>Re Labeled DNA).
This method is greatly used in the analysis of copy number variants (CNVs) in genes. CGH is widely used in Cancer diagnosis and research as well as to study evolutionary changes in the human genome by carrying out CGH using ancestral DNA (Ex. Chimpanzee) with Human DNA.
Advantages
- 1) Highly sensitive method can detect minor changes in copy number variations (CNVs.)
- 2) A screening method that can screen the entire genome for CNVs.
Disadvantages
- 1) Whole genome labeling is expensive.
- 2) Can detect only gain/ loss in the sample’s DNA but can not detect any other type of aberration
3.6.4 Array CGH (aCGH)
DNA microarrays allow for simultaneous analysis of genes products or DNA copy numbers for thousands of loci. These microarrays contain defined human chromosomal segments (isolated from a chromosomal library), that have been ‘spotted’ or fixed onto a glass slide. The spotting is done in a very precise manner such that the location of each spot as well its content is clearly known. The microarray will contain several hundreds of such ‘spots’. Once this is complete, then standard CGH protocol will be followed to determine gain or loss of genetic material for all the spots on the microarray. The microarray is then incubated with the labeled test and control DNA. The array is then washed to remove DNA that is not bound, and the positions in the microarray with labeled DNA fragments.
The resolution that can be achieved with this type of analysis is greater than what can be done with conventional CGH. This is because the spots on the microarray can have a defined size. In addition to this the analysis can be done for Single Nucletide Polymophism which can be mapped using aCGH. However, the resolution of SNP arrays is currently limited to about 10,000 SNPs per chip.
Advantages
- 1) Highly sensitive, can carry out analysis for several thousands of variants simultaneously.
- 2) CNV as well as SNP analysis can be done.
Disadvantages
- 1) Very costly to carry out due to high cost of array production and analysis.
- 2) Low reproducibility of tests.
3.7 FLOW KARYOTYPING
Flow Cytometry is a method that is used to distinguish and separate particles such as cells or chromosomes. The mechanism involved uses the combinations of fluorescent labels to create unique labels for the desired chromosomes. Thechromosomes in a suitable buffer are then passed through a narrow nozzle of the flowcytometer. The nozzle is so narrow that each droplet will contain only a few chromosomes. The drops are then scanned with a laser beam that excites the flours present. The fluorescent signals are then picked up by a detection system; the signal is then amplified and based in the presence or absence of a particular signal the drop is charged using a charging ring. This charged droplet is deflected using charged deflection plates into suitable collection vials. In this method the sorting can be done based on the type of signal being observed as well as the strength of the signal. The overview of the method has been represented in Fig. 3.11.
One of the most common methods is the use of two different dyes. One is, called Hoechst 33269, binds to A-T base pairs of DNA the other is Chromomycin A which binds to the G-C rich regions. This combination can be used to sort all the chromosomes based on the principle that larger the chromosome, greater will be the AT and GC content, the greater this content the greater will be the resultant signal strength. Using the strength of the signal the chromosomes can be sorted.
As illustrated in Figure 3.11, the smallest chromosome i.e. 21 shows the lowest Hoechst and chromomycin staining intensity. Chromosomes 1 and 2, which are the largest, show the highest Hoechst and chromomycin staining.
Advantages
- 1) Highly sensitive, capable of carrying out analysis in real time.
- 2) High throughput, large number of sample can be analyzed.
Disadvantages
- 1) Trained personnel required to handle instrument.
- 2) Currently can only detect numerical aberration accurately, used most commonly to determine ploidy in samples.
Chromosomes that are released from mitotic cells are stained with two DNAbinding dyes with different base-pair specificities, and the fluorescence intensities of each of several thousand chromosomes are measured in a two-laser flow cytometer. In the example shown, the two dyes are Hoechst 33258, which binds preferentially to A-T base pairs, and chromomycin A3, which binds to C-G base pairs. The resulting bivariate “flow karyotype” (bottom right panel) resolves all chromosomes except for the 9–12 group. In this example, maternal and paternal homologous of both chromosomes 21 and 19 are resolved into separate peaks owing to differences in their DNA content. After measurement, droplets that contain desired chromosomes, such as chromosome 3 in this example can be deflected into tubes for molecular analyses. UV, ultraviolet
Sample Questions
- 1) What is a Karyotype?
- 2) Describe the medium most commonly used for Lymphocyte Culture.
- 3) What are the drawbacks in conventional karyotyping?
- 4) Describe different Chromosomal banding techniques.
- 5) What is FISH? What are the different types of Probes used in FISH analysis
- 6) What is SKY? Describe its applications, advantages and disadvantages.
- 7) Describe in detail the methods of CGH and aCGH.
- 8) What is Flow Karyotyping?