CHROMOSOME ANALYSIS

NARENDRA YADAV

Msc II

PAPER II (MEDICAL MICROBIOLOGY)

WHAT ARE CHROMOSOMES??

In the nucleus of each cell, the DNA

molecule is packaged into thread-like

structures called chromosomes. Each

chromosome is made up of DNA tightly coiled

many times around proteins called histones

that support its structure.

Humans have 46 chromosomes, or 23 pairs

The first 22 pairs are called autosomes.

The 23rd pair are the sex chromosomes OR

gonosomes – this pair will either be XX or

XY.

INTRODUCTION

Chromosome analysis is a test that

evaluates the number and structure of

a person's chromosomes in order to

detect abnormalities.

Chromosomal abnormalities include both

numerical and structural changes.

For numerical changes, anything other than a complete set of 46

chromosomes represents a change in the amount of genetic material

present and can cause health and development problems. For structural

changes, the significance of the problems and their severity depends

upon the chromosome that is altered.

TECHNIQUES FOR

CHROMOSOME ANALYSIS

1.Fluorescent In-situ Hybridization (FISH)

2.Comparative genomic hybridization (CGH)

3.Array Comparative Genomic Hybridization (ARRAY CGH)

CHROMOSOME ANALYSIS

Chromosome analysis has to be performed

on dividing cells.

1.Peripheral blood lymphocytes.

2.Amniotic fluid.

3.Cultured skin fibroblasts.

4.Bone marrow cells.

FISH(Fluorescent in situ hybridization)

Fluorescent In-situ Hybridization

(FISH) is a technique which uses a

fluorescently-labeled probe to detect

the presence or absence of a

particular chromosome segment or gene.

This technique can detect small

deletions, duplications and/or subtle

chromosomal rearrangements.

PROCEDURE

• The slide is aged using a salt solution usually consisting of 2X SSC (salt, sodium citrate).

• The slides are then dehydrated in ethanol, and the probe

mixture is added.

• The sample DNA and the probe DNA are then co-denatured using

a heated plate and allowed to re-anneal for at least 4

hours.

• The slides are then washed to remove excess unbound probe,

and counterstained with 4',6-Diamidino-2-phenylindole

(DAPI).

DENATURE OF DNA

DENATURE OF PROBE

LABELLING WITH PROBE

HYBRIZATION OF DNA-PROBE

DETECTION

ANALYSIS

Analysis of FISH specimens is done

by fluorescence microscopy by a clinical

laboratory specialist in cytogenetics.

For congenital problems usually

20 metaphase cells are scored.

Examples of diseases that are diagnosed using FISH include Prader-Willi

syndrome, Angelman syndrome, 22q13 deletion syndrome, chronicmyelogenous

leukemia, acute lymphoblastic leukemia, Cri-du-chat, Velocardiofacial

syndrome and Down syndrome.

DIFFERENT TYPES OF FISH

• Stellaris® RNA FISH.

• Fiber FISH.

• Q-FISH.

• Flow-FISH.

• M-FISH

Comparative genomic

hybridization (CGH)

Comparative genomic hybridization is a molecular cytogenetic method

for analysing copy number variations (CNVs) relative

to ploidy level in the DNA of a test sample compared to a reference

sample.

The aim of this technique is to quickly and efficiently compare two

genomic DNA samples arising from two sources, which are most often

closely related, because it is suspected that they contain

differences in terms of either gains or losses of either

whole chromosomes or subchromosomal regions (a portion of a whole

chromosome).

PROCEDURE

This Technique involves the isolation of DNA from the two sources to be

compared.

Most commonly a test and reference source.

Independent labelling of each DNA sample with a

different fluorophores (fluorescent molecules) of different colours

(usually red and green).

Denaturation of the DNA so that it is single stranded, and

the hybridization of the two resultant samples in a 1:1 ratio to a

normal metaphase spread of chromosomes, to which the labelled DNA samples

will bind at their locus of origin.

REFERENCE CELL (NORMAL)

TEST CELL

1234567

DYE

HYBRIDIZATION

PROCEDURE

GRAPH

0.75 1 1.25

REFERENCE CELL (NORMAL)

TEST CELL (DISEASED)

1234567

DYE

HYBRIDIZATION

PROCEDURE

GRAPH

DUPLICATION

0.75 1 1.25

REFERENCE CELL (NORMAL)

TEST CELL

1234567

DYE

HYBRIDIZATION

PROCEDURE

GRAPH

DELETION

0.75 1 1.25

ADVANTAGE & LIMITATION

CGH is only able to detect

unbalanced chromosomal abnormalities.

Conventional CGH has been used mainly for

the identification of chromosomal regions

that are recurrently lost or gained in

tumors.

A main disadvantage of conventional CGH is

its inability to detect structural

chromosomal aberrations without copy number

changes.

for eg. balanced chromosomal

abnormalities such as reciprocal

translocations, or inversions do not

affect copy number.

Array Comparative Genomic

Hybridization (ARRAY CGH)

• Array comparative genomic hybridization (also microarray-based comparative genomic hybridization, matrix CGH, array CGH, aCGH) is a molecular cytogenetic technique for the detection of chromosomal copy number changes on a genome wide and high-resolution scale.

• With the introduction of array CGH, the main limitation of conventional CGH, a low resolution, is overcome.

• Using this method, copy number changes at a level of 5–10 kilobases of DNA sequences can be detected.

ADVANTAGE & LIMITATION

• Aberrations smaller than 5–10 Mb cannot be detected using conventional CGH. For the detection of such

abnormalities, a high-resolution technique is required.

Array CGH overcomes many of these limitations.

• The main disadvantage of array CGH is its inability to

detect aberrations that do not result in copy number

changes.

APPLICATION

1.Conventional CGH

A. CGH in cancer research

CGH data from several studies of the same tumor type show

consistent patterns of non-random genetic aberrations.

For example, 13q gain 9q loss in bladder cancer, 14q loss in renal

cancer.

B.Chromosomal Aberrations.

• Cri du Chat (CdC) is a syndrome caused by a partial

deletion of the short arm of chromosome 5.

• Several studies have shown that conventional CGH is

suitable to detect the deletion, as well as more

complex chromosomal alterations.

2.Array Comparative Genomic Hybridization (ARRAY

CGH)

A.Submicroscopic aberrations

• Prader–Willi syndrome (PWS) is a paternal structural abnormality

involving 15q11-13, while a maternal aberration in the same region

causes Angelman syndrome (AS).

• In both syndromes, the majority of cases (75%) are the result of

a 3–5 Mb deletion of the PWS/AS critical region. These small

aberrations cannot be detected using cytogenetics or conventional

CGH, but can be readily detected using array CGH.

• Array CGH applications are mainly directed at detecting genomic abnormalities in cancer.

• However, array CGH is also suitable for the analysis

of DNA copy number aberrations that cause human genetic

disorders.

• https://en.wikipedia.org/wiki/Comparative_genomic_hybridization#Array_Comparative_Genomic_Hybr

idization

• http://www.utmb.edu/pedi_ed/CORE/MedicalGenetics/page_09.htm

• https://en.wikipedia.org/wiki/Fluorescence_in_situ_hybridization#Preparation_and_hybridization

_process_.E2.80.93_DNA

REFRENCE