Lectures Clinical Genetics

1. Lectures Clinical Genetics Dr. AneelaJaved

2. Chapter 5: How to study patients DNA Two essentials 1- hybridization 2-Amplification of sequence of interest 1- hybridization for DNA testing in dif techniques:labeling with fluorescent probes Dot blot: point mutation or small seq changesickle cell (SCA): point mutation in the β-globin chain of haemoglobin, hydrophilic amino acid glutamic acid hydrophobic amino acid valine The β-globin gene is found on chromosome 11

4. Southern blotting: Large scale changes, inversions, deletions, that change pattern of restriction fragments, trinucleotide repeat expansions that are too large to amplified by PCR.

7. Fragile X syndrome expansion of the CGG trinucleotide repeat at 5’utr, affecting the Fragile X mental retardation 1 (FMR1) gene on the X chromosome, required for normal neural development. length of the CGG repeat, 55 (unaffected by the syndrome), above 55 unstable a premutation (at risk of fragile X associated disorders), or full mutation 200 or > (usually affected by the syndrome). As gc repeats are difficult to amplify or detect by pcr so Southern blottB, x –inactivation of repeat expansions cause methylation of FMR1 promotor thus restriction enzyme ECLx 1 is unable to cut individuals with FXS due to missense mutations or deletions involving FMR1 will not be diagnosed using this test and should therefore undergo sequencing of the FMR1 gene if there is clinical suspicion of FXS.

8. FISH • FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence complementarity. • Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes. • Chk the presence or absence of seq, copy number and chromosomal location of seq at least several kb long. or presence or absence of chromosomes.

9. Deletions: probe will not bind Change in chr no: down syndrome chromosome specific probe

10. First, a probe is constructed. The probe 1okb or so 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. • an interphase or metaphasechromosomes are denatured and firmly attached to a substrate, usually glass. • The probe is then applied to the chromosome DNA and incubated for approximately 12 hours while hybridizing. • Several wash steps remove all unhybridized or partially hybridized probes. • The results are then visualized and quantified using a microscope that is capable of exciting the dye and recording images. • If the fluorescent signal is weak, amplification of the signal may be necessary in order to exceed the detection threshold of the microscope. • Fluorescent signal strength depends on many factors • probe labeling efficiency, • the type of probe, • and the type of dye.

11. Different types of FISH Probes • Centromeric Probes; consist of repetitive DNA sequences found in and arround the centromere of a specific chromosomes. • Used for rapid diagnosis of trisomies 13, 18, 21. • 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(21q22.13-21q22.2), X and Y chromosomal abnormalities. • Telomeric probes; Complete set of telomeric probes for all 24 chromosomes, used for subtelomeric abnormalities (deletions, translocations). • Whole chromosome paint probes; consist of a cocktail of probes obtained from different parts of a particular chromosome, used for ring chromosomes and translocations.

14. M-FISH. Multiplex in situ hybridization (M-FISH) is a 24-color karyotyping technique for studying complex interchromosomal rearrangements. The process involves three major steps. Firstly, the multiplex labeling of all chromosomes in the genome with finite numbers of spectrally distinct fluorophores in a combinatorial fashion, such that each homologous pair of chromosomes is uniquely labeled. Secondly, the microscopic visualization and digital acquisition of each fluorophore using specific single band-pass filter sets and dedicated M-FISH software. These acquired images are then superimposed enabling individual chromosomes to be classified based on the fluor composition in accordance with the combinatorial labeling scheme of the M-FISH probe cocktail used. The third step involves the detailed analysis of these digitally acquired and processed images to resolve structural and numerical abnormalities.

15. Figure 3: Spectral karyotyping and multifluor FISH paint each human chromosome in one of 24 colors.

16. uses mixtures of differently colored probes. • A range of colors in mixtures of fluorescent dyes can be detected, so each human chromosome can be identified by a characteristic color using whole-chromosome probe mixtures and a variety of ratios of colors. • Although there are more chromosomes than easily distinguishable fluorescent dye colors, ratios of probe mixtures can be used to create secondary colors. the probe mixture for the secondary colors is created by mixing the correct ratio of two sets of differently colored probes for the same chromosome.

17. FIBER FISH interphase chromosomes are attached to a slide in such a way that they are stretched out in a straight line, rather than being tightly coiled, as in conventional FISH, This is accomplished by applying mechanical shear along the length of the slide, either to cells that have been fixed to the slide and then lysed, or to a solution of purified DNA. A technique known as chromosome combing is increasingly used for this purpose. The extended conformation of the chromosomes allows dramatically higher resolution - even down to a few kilobases. The preparation of fiber FISH samples, although conceptually simple, is a rather skilled art, and only specialized laboratories use the technique routinely.

19. allows the visualization of individual genes or other small DNA elements on chromosomes with a resolution of approx 1000 bp. Applications of fiber-FISH range from the determination of numbers of repetitive genes to establishing the physical order of cloned DNA fragments along continuous sections of individual chromosomes. Particularly in organisms with relatively small and gene dense genomes, such as protozoan parasites, fiber-FISH can easily be used as a complementary technique to classical in vitro mapping approaches.

20. Q-FISH Q-FISH combines FISH with PNAs and computer software to quantify fluorescence intensity. This technique is used routinely in telomere length research. labelled (Cy3 or FITC) syntheticDNA mimics  peptide nucleic acid (PNA) oligonucleotides .

22. FLOW-FISH Flow-FISH uses flow cytometry to perform FISH automatically using per-cell fluorescence measurements.