1. Chromosomes as functioning organelles Telomeres
Telomeres are specialized structures, comprising DNA and protein, which cap the ends of eukaryotic chromosomes.
Functions
Maintaining the structural integrity of a chromosome.
Ensuring complete replication of the extreme ends of chromosomes.
Helping establish the three-dimensional architecture of the nucleus and/or chromosome pairing.
The ability of telomerase to replicate a chromosome end depends on the unique molecular structure of the telomere.

2. Chromosomes as functioning organelles Telomeres
Eukaryotic telomeres consist of a long array of tandem repeats. One DNA strand contains TG-rich sequences and terminates in the 3′ end; the complementary strand is CA-rich.
Highly conserved in evolution - there is considerable similarity in the simple sequence repeat,
Example
TTGGGG (Paramecium), TAGGG (Trypanosoma) TTTAGGG (Arabidopsis) and TTAGGG (Homo sapiens)

3. Chromosomes as functioning organelles Telomeres
First isolated from the protozoan Tetrahymena thermophila and possess multiple copies of the sequence:

4. Human Telomeres
The (TTAGGG)" array of a human telomere spans about kb.
A very large protein complex shelterin, or the telosome contains several components that recognize and bind to telomeric DNA.
Two telomere repeat binding factors (TRFl and TRF2) bind to double-stranded TTAGGG sequences.
G-rich strand has a Single-stranded overhang at its 3' end that is typically nucleotides long.
This can fold back and form base pairs with the other, C-rich, strand to form a telomeric loop known as theT-loop.
Protect the telomere DNA from natural cellular mechanisms that repair double-stranded DNA breaks.

6. Chromosomes as functioning organelles Telomeres
Telomeres have now been isolated from protozoans, plants, humans, and other organisms; most are similar in structure.

7. Chromosomes as functioning organelles Telomere Structure
The G-rich strand often protrudes beyond the complementary C-rich strand at the end of the chromosome.
The length of the telomeric sequence varies from chromosome to chromosome and from cell to cell, suggesting that each telomere is a dynamic structure that actively grows and shrinks.
The telomeres of Drosophila chromosomes are different in structure.
They consist of multiple copies of the two different retrotransposons , Het-A and Tart, arranged in tandem repeats.
Apparently, in Drosophila, loss of telomere sequences during replication is balanced by transposition of additional copies of the Het-A and Tart elements.

8. Telomerase and the chromosome end-replication problem

9. MOLECULAR CYTOGENETIC TECHNIQUES
Dr Attya Bhatti

10. Molecular Methods for chromosomal analysis Molecular Cytogenetics
Fluorescent in situ Hybridization (FISH)
Chromosome painting
Comparative Genomic Hybridization (CGH)
Molecular karyotyping and Multiplex FISH(M-FISH)
Spectral Karyotyping
Array CGH

11. In situ hybridization
In situ hybridization, DNA probes can be used to determine the chromosomal location of a gene or the cellular location of a mRNA in a process called in situ hybridization.
The name is derived from the fact that DNA or RNA is visualized while it is in the cell (in situ).
High resolution of conventional FISH on metaphase chromosomes is several mega bases.
Prometaphase chromosomes can permit 1 Mb resolution.
For higher resolution:
naturally extended interphase chromosomes (50 – 500kb range)
Artificially extended chromosomes/DNA fibers
Artificially extended chromosomes can be obtained by procedures like DIRVISH (DIRect VISual Hybridization) which involves lysing cells with detergent at one end of a glass slide, tipping the slide , allowing the DNA solution to stream down the slide. Such preparations permit extremely high resolution. ( over 700 kb to under 5 kb )

12. Fluorescent IN SITU Hybridization (FISH)
A technology used to
Detect & localize the presence/absence of specific DNA sequences on chromosomes
for finding specific features in DNA( medicine, and species identification)
detect and localize specific RNA targets (mRNA, lncRNA and miRNA)
Principle :
Labeled nucleic acid sequence/ probes are used for the visualization of specific DNA or RNA sequences on mitotic chromosome preparations or in interphase cells. Probe construction:
Probes large enough to hybridize specifically but not so large to impede hybridization
Tagged directly with fluorophores, with targets for antibodies or with biotin.
Tagging can be done in various ways, such as nick translation, or PCR using tagged nucleotides
Concept: A simple procedure for mapping genes and other DNA sequences is to hybridize a suitable labeled DNA probe against chromosomal DNA that has been denatured in situ.
First, a probe is constructed. The probe must be large enough to hybridize specifically with its target but not so large as to impede the hybridization process. 
If the fluorescent signal is weak, amplification of the signal may be necessary in order to exceed the detection threshold of themicroscope. Fluorescent signal strength depends on many factors such as probe labeling efficiency, the type of probe, and the type of dye. Fluorescently tagged antibodies or streptavidin are bound to the dye molecule. These secondary components are selected so that they have a strong signal.
Cells, circulating tumor cells (CTCs), or formalin-fixed paraffin-embedded (FFPE) or frozen tissue sections are fixed, then permeabilized to allow target accessibility. FISH has also been successfully done on unfixed cells.[10] A target-specific probe, composed of 20 oligonucleotide pairs, hybridizes to the target RNA(s
First, a probe is constructed. The probe must be large enough to hybridize specifically with its target but not so large as to impede the hybridization process. The probe is tagged directly with fluorophores, with targets for antibodies or with biotin.
Tagging can be done in various ways, such as nick translation, or PCR using tagged nucleotides.
Nick translation [1] (or head translation), developed in 1977 by Rigby and Paul Berg, is a tagging technique in molecular biology in which DNA Polymerase I is used to replace some of the nucleotides of a DNA sequence with their labeled analogues, creating a tagged DNA sequence which can be used as a probe in Fluorescent in situ hybridization orblotting techniques. It can also be used for radiolabeling.

13. The sample DNA (metaphase chromosomes or interphase nuclei) is first denatured, a process that separates the complimentary strands within the DNA double helix structure.
The fluorescently labeled probe of interest is then added to the denatured sample mixture and hybridizes with the sample DNA at the target site as it reanneals (or reforms itself) back into a double helix.
The probe signal can then be seen through a fluorescent microscope and the sample DNA scored for the presence or absence of the signal.

14. Different types of FISH Probes
1. Centromeric Probes; consist of repetitive DNA sequences found in and around the centromere of a specific chromosomes.
Used for rapid diagnosis of trisomies 13, 18, 21.
2. Chomosomes specific unique sequence probes; specific for a particular single locus. Locus specific probes for chromosomes 13q14 and the critical region for down syndrome on chr.21(21q q22.2), X and Y chromosomal abnormalities.
3. Telomeric probes; Complete set of telomeric probes for all 24 chromosomes, used for subtelomeric abnormalities (deletions, translocations).
4. Whole chromosome paint probes; consist of a cocktail of probes obtained from different parts of a particular chromosome, used for ring chromosomes and translocations.

15. Chromosomal painting
Use of DNA probes where starting DNA is composed of a large collection of different DNA fragments from a single type of chromosome.
Chromosome specific DNA library used for this purpose
Hybridization signal generated represents combined contribution of many loci spanning a whole chromosome and causes whole chromosome to fluoresce.
Limitation: Availability of lesser # of fluorescent dyes.
Solutions: A.) Combinatorial labeling
B.) Ratio labeling
Automated digital image analysis instead of fluorescence microscopy.
The long-awaited goal of simultaneously visualizing all 24 different human chromosomes achieved via 2 approaches.
Multiplex FISH (M-FISH)
Spectral karyotyping(SKY)
Combinatorial labeling: Labeling individual probes with more than one type of fluorophore.
Ratio labeling: Using a combination of different fluorophores but also, in different ratios.

16. Approaches and Applications
M-FISH: Uses digital images acquired separately for each of 5 different fluorophores using a CCD camera. Images are then analyzed by a software that generates a composite image in which each chromosome is given a different pseudocolor depending on the fluorophore composition.
SKY: CCD imaging is combined with Fourier spectroscopy. The spectrum of fluorescent wavelengths for each pixel is assessed using an interferometer and a dedicated computer program assigns a specific pseudocolor depending on particular fluorescence spectrum identified.
Applications:
De-novo rearrangements and marker chromosome definition in clinical and cancer cytogenetics.
1. Chromosomal preparations from tumors are of poor quality
2. Complex chromosomal rearrangements are frequent in tumor samples.

17. Different types of Fish Probes
5. Probes derived from flow- sorted chromosomes; Because of their size and DNA composition, chromosomes bind different amount of fluorescent dyes, some of which bind specifically to GC sequences and others to AT sequences.
This property of differential binding allows chromosomes to be separated by the process of flow cytometry or fluorescent activated cell sorting (FACS). This will stain the metaphase chromosomes with a florescent DNA- binding dye and then project them across a laser beam which excites the chromosome to fluoresce. This fluorescence intensity is measured and analyzed by a computer that draws up a distribution histogram of chromosomes size called as a flow karyotype.

18. Spectral Karyotyping

19. WHAT IS SPECTRAL KARYOTYPING
Spectral karyotyping (SKY) is a laboratory molecular cytogenetic technique that allows scientists to visualize all pairs of chromosomes in an organism at one time by "painting" each pair of chromosomes in a different fluorescent color
Based on the approach of the fluorescence in situ hybridization technique

20. WHAT IS SKY USED FOR…?
Many diseases are associated with particular chromosomal abnormalities. For example, chromosomes in cancer cells frequently exhibit aberrations called translocations, in which a piece of one chromosome breaks off and attaches to the end of another chromosome. Identifying such chromosomal abnormalities and determining their role in disease is an important step in developing new methods for pre- and postnatal diagnostics in cancer and other genetic disorders.
Traditional karyotyping allows scientists to view the full set of human chromosomes in black and white, a technique that is useful for observing the number, size and shape of the chromosomes. Interpreting these karyotypes, however, requires an expert, who might need hours to examine a single chromosome. By using SKY, even non-experts can easily see instances where a chromosome, painted in one color, has a small piece of a different chromosome, painted in another color, attached to it. Combinatorial pseudocolors are assigned to each segment.

21. HOW DOES SKY WORK…??
PROBES: SKY involves the preparation of a large collection of short sequences of single-stranded DNA called probes. Each of the individual probes in this DNA "library" is complementary to a unique region of one chromosome - together, all of the probes make up a collection of DNA that is complementary to all of the chromosomes within the human genome.
FLUOROPHORES: Each probe is labeled with a fluorescent color (fluorophores) that is designated for a specific chromosome.
SPECTRAL ANALYSIS: When these probes are mixed with the chromosomes from a human cell, the probes hybridize - bind - to the DNA in the chromosomes. As they hybridize, the fluorescent probes essentially paint the full set of chromosomes in a rainbow of colors. Scientists can then use computers to analyze the painted chromosomes to determine whether any of them exhibits translocations or other structural abnormalities.

22. INTERFEROMETER:
A unique feature of the technology is the use of an interferometer attached to a fluorescence microscope. Slight variations in color, undetectable by the human eye, are detected by a computer program that then reassigns an easy-to-distinguish color to each pair of chromosomes. The result is a digital image rather than film, in full color. Pairing of the chromosomes is simpler because homologous pairs are the same color, and aberrations and cross-overs are more easily recognizable.

24. Applications
Spectral Karyotyping (SKY) can detect 1. Chromosomal material of unknown origin 2. Complex rearrangements 3. Translocations 4. Large deletions 5. Duplications 6. Aneuploidy
Pre- and postnatal characterization of certain numerical and structural rearrangements and complex karyotypes
Highly informative analysis of sample materials with only single or few cells available for investigation

25. CGH and Array CGH

26. Comparative Genomic Hybridization
Cytogenetic technique for genome wide screening of copy number variations
Principle??
The principle of this technique is to efficiently compare two genomic DNA samples arising from two sources and determine differences in terms of gain or loss of either whole chromosomes or sub- chromosomal regions
Why perform CGH?
No prior knowledge of the region required
Unlike other techniques scan entire genome
No need of dividing cells
Detect submicroscopic alterations

28. Array CGH

30. Applications of CGH
Simultaneously detect aneuploidies, deletions, duplications and amplification of any locus
Chromosomal aberrations
Cancer research
Prenatal genetic diagnosis