CYTOGENETICS

CYTOGENETICS Study of chromosomes and related

diseases caused by abnormality in its structure and number

Study of concepts related to the study of heredity, genetic phenomenon, sex determination and disorders in relation to human inheritance.

HUMAN G E N E

Segment of DNA that codes for a polypeptide.

CHROMOSOMES – the three-dimensional structure within the nucleus of the cell made up of a continuous molecule of DNA and proteins wrapped around it.

CHROMATIN – composed mainly of coiled strands of DNA and its associated histone protein . They represent chromosomes in different degrees of uncoiling

COMPONENTS OF CHROMATIN

1. DNA2. Histones3. DNA binding proteins4. RNA

TYPES OF CHROMATIN

HETEROCHROMATIN EUCHROMATIN1. Transcriptionally

inactive2. Highly condensed form

3. Located near centromeres & telomeres

4. Makes up 10% of the chromatin present in an interphase cell

5. DNA replicated late in S phase

1. Transcriptionally active

2. Less condensed ,loosely packed

3. Located near the nuclear pore complex

4. Comprises 90% of chromatin in a interphase cell

5. DNA replicated early in S phase

HUMAN DNA CONTAINS TWO COPIES OF EVERY DNA SEQUENCE; IS ORGANIZED INTO CHROMOSOMES W/C SERVE TO MANEUVER DNA THROUGH CELL DIVISION

ESSENTIAL PARTS OF A CHROMOSOMES

1. TELOMERES – Chromosome tips containing many repeats of the sequence TTAGGG.

2. CENTROMERES- largest constriction of a chromosome ; the site where spindle fibers attach.

3. ORIGIN OF REPLICATION SITES – where replicaton fork begin to form

HOW IS DNA CONDENSED INTO A CHROMOSOMES ?

By wrapping around histone proteins and forms compressed loops of DNA

CLASSIFICATION OF CHROMOSOMES

1. TELOCENTRIC - has the centromere at one end

2. ACROCENTRIC - has the centromere near an end of chromosomes

3. SUBMETACENTRIC – centromere creates a long q arm and a short p arm

4. METACENTRIC – centromere establishes equal sized p and q arms.

TYPES OF CHROMOSOMES

A. METACENTRIC B. SUBMETACENTRICC. ACROCENTRICD. TELOCENTRIC

KARYOTYPE

Standard chart of chromosomesisolated from a cell at metaphase arranged in order by size and structure or physical landmark

SPECIMENS USED FOR OBTAINING CHROMOSOMES PREPARATION :

1. Peripheral blood 2. Bone Marrow3. Cultured skin fibroblasts4.Amniotic fluid5. Products of Conception – Chorionic villi

PROTOCOLS FOR KARYOTYPING

1. A sample of blood is drawn and placed in a tube containing heparin as anticoagulant.

2.Through density gradient centrifugation technique, mononuclear cells are isolated are isolated and purified

3. Mononuclear cells are then cultured for 3-4 days in a medium containing phytohemagglutinin.

4. At the end of the culture period, the large population of dividing cells is treated with a drug e.g. Colcemid.

PROTOCOLS FOR KARYOTYPING

5. The lymphocytes are harvested and treated briefly with a hypotonic solution

6.The swollen cells are fixed, dropped onto a microscope slides, dried and stained to induce a banding pattern .

7. Stained slides are scanned to identify good chromosome spread and they are then photographed.

8. Photos showing images of each chromosomes are cut out and pasted to a backing sheet in an orderly manner.

PROTOCOLS FOR KARYOTYPING

CONSTRUCTING A KARYOTYPE

The stained metaphase chromosomes preparations are examined microscopically with 15-20 cells scanned and counted. At least five cells are fully analyzed.

In doing a full analysis each chromosome is critically compared band-for-band with its homolog.

BANDING TECHNIQUES

1. Q BANDING – chromosomes are stained with quinacrine

2. G BANDING – chromosomes are first digested with trypsin then stained with Giemsa

3. C BANDING – chromosomes are treated with acid & base solutions then stained with Giemsa

4. R BANDING – Uses olivomycin or acridine orange

HUMAN MALE & FEMALE CHROMOSOMES

IDEOGRAM

A simple cartoon of a chromosomes, often used in genomics to show the over-all physical structure of a chromosomes

IDEOGRAM

CLASSIFICATION OF CHROMOSOMES FOR KARYOTYPING

GROUP A – Chromosomes 1-3 , largest, metacentric chromosomes

GROUP B – Chromosomes 4-5, large with submedian centromeres

GROUP C –chromosomes 6-12, medium sized with sub-median centromere

GROUP D – Chromosomes 13-15, medium sized with acrocentric centromere

GROUP E – Chromosomes 16-18, short with median or submedian centromere

GROUP F – chromosomes 19-20, short with median centromere

GROUP G- chromosomes 21-22, very short with acrocentric centromere

CHROMOSOME X similar to Group CCHROMOSOME Y similar to Group G

CLASSIFICATION OF CHROMOSOMES FOR KARYOTYPING