Practical Of Genetics

Transcription

1 Practical Of Genetics

2 1. Students will be able to demonstrate a microtechnique for reliable chromosomal analysis of leucocytes obtained from peripheral blood. 2. Students will be able to prepare a karyotype from the chromosomes of a normal human male or female. 3. Students will be able to use the karyotyping techniques for diagnosing a chromosomal disorder.

3

4

5 Structure Number Deletion Insertion Duplication Translocation Inversion Aneuploidy Euploidy

6 are abnormalities in the number or microscopically observable structure of chromosomes. The normal human diploid cell has 23 pairs of chromosomes visible only at the metaphase stage of mitosis, 22 homologous pairs of autosomes and two sex chromosomes.

7

8

9 Triploidy

10

11 Trisomy 13: Patau Syndrome (47,+13) Trisomy 18: Edwards Syndrome (47,+18) Trisomy 21: Down Syndrome (47,+21)

12

13 Klinefelter s XXY Turner s X0 XYY Syndrome XYY

14

15

16 XYY Syndrome: 47, XYY

17 Detecting Cancer

18

19 Each chromosome has a characteristic and in the normal cell. Chromosome pair contains the largest chromosomes, while the chromosome is the smallest. Chromosomes of are grouped together by letter; the chromosomes can be arranged in 7 groups ( ). Thus group A contains pairs #1, #2, and #3 chromosomes.

20 A chromosome is divided by its centromer into short arm p (= petite or short) and long arm q (= queue, or long) p q

21 Chromosomes can be classified by the position of their centromer: Metacentric: Submetacentric:. Acrocentric:

22

23 Telocentric

24

25 A 1-3 Large metacentric B C 4,5 Large submetacentric 6-12, X Medium submetacentric D medium acrocentric E F G short metacentric Short metacentric 21,22,Y Short acrocentric

26

27

28

29 Divided into two arms by the centromere Each arm subdivided into bands, numbered from the centromere outwards Any given point on a chromosome is designated by the chromosome number, arm, arm region and the band 15q12 Xp11 Chromosome number: X; Arm: p; Region: 1; Band: 1

30 is a technique that allows for the visualization and identification of human metaphase chromosomes. This technique can be used to assess the normalcy of an individual s chromosomes and to assay for various genetic diseases such as Down s syndrome and Klinefelter s syndrome etc.

31 Samples for chromosomal analysis can be prepared relatively easily using skin, bone morrow, chorionic villy or cells from amniotic fluid.

32

33

34 It is estimated that one in 156 live births have some kind of chromosomal abnormality. To create a karyotype, chromosomes from a cell are arranged, stained and photographed. The photograph is enlarged and cut up into individual chromosomes. The homologous chromosomes can be distinguished by length and by the position of the centromer.

35 A blood sample is taken and white blood cells grown in special medium for three days under the influence of the ( ) to enter into mitosis by DNA replication.

36 After hours, ( ) is added to the culture to stop mitosis in the metaphase stage. After treatment by ( ) to causes a swelling of the cells and allow dispersion of the chromosomes within the cell membrane.

37 is used as wash solution to lyse the remeaning red cells and remove some chromosomes protein. After, chromosomes can be microscopically observed and evaluated for abnormalities.

38 Metirals Heparinzed whole blood Heparin sodium injection Peripheral blood Karyotyping medium with PHA (RPMI 1640) Incubator 5% CO 2 at 37 C Colcemide solution 10 g/ml M KCl Fixative solution ( 3x methanol : 1x glacial acetic acid ) Giemsa stain solution Slides and Microscope

39 Blood culture media; 500 ml RPMI 1640 with 100ml fetal bovine serum, 6.5ml penicillin streptomycin and 7ml glutamine. Dispense 10ml aliquots into sterile tube and add 2% (0.2ml) PHA to each tube. Store at 4 C for along as 2 weeks.

40

41

42 - Condensed chromatin and genetically inactive (limited transcription) during interphase. - It consists of repetitive DNA sequences which are relatively rich in AT base pairs and is late replicating in the cell cycle. - Is darkly stained. - Heterochromatin is believed to serve several functions, from gene regulation to the protection of the integrity of chromosomes.

43 - Chromatin that is not belonged to heterochromatin is euchromatin. - Is lightly stained due to the less compact structure. - Early-replicating and GC rich region. - It should be noted that in prokaryotes, euchromatin is the only form of chromatin present. - Euchromatin participates in the active transcription of DNA to mrna products.

44 - Chromosome banding is developed based on the presence of heterochromatin and euchromatin. - Heterochromatin is darkly stained whereas euchromatin is lightly stained during chromosome staining. - A band is defined as that part of a chromosome which is clearly distinguishable from its adjacent segments by appearing darker or brighter with one or more banding techniques. - There are a few types of chromosome banding: g- banding, c-banding, q-banding, r-banding etc.

45 Giemsa stain is used in Giemsa banding, commonly called G-banding, Used to produce a pattern of bands specific to each type of chromosome One common method is G-banding Treated with trypsin Stained with Giemsa stain

46 - G-banding is obtained with giemsa stain following digestion of chromosomes with enzyme trypsin. - It yields a series of lightly and darkly stained bands - the dark regions tend to be heterochromatic, late-replicating and at rich. The light regions tend to be euchromatic, earlyreplicating and GC rich.

47 G-Banding G-banding of human female metaphase chromosomes

48 Karyotyping procedure 1- Inoculate 0.5ml of heparinized whole blood into tube with 10ml of karyotyping medium. 2- Incubate the tubes in incubator with 5% CO 2 at 37 o C for total of 72 hours. 3- After total of 69 hours from seeding add 100μl of Colcemid Solution to each culture tubes. 4- Incubate the tubes at 37 o C for an additional minutes. 5- Spin at 500g (1500 rpm) for 7 minutes. 6-Remove the supernatant and re-suspend the cells in 5ml of hypotonic 0.075M KCl prewormed to 37 o C.

49 7- Incubate at 37 o C for 15 minutes. 8- Add drop-by- drop (with vortexing) 1ml fresh ice cold fixative. 9- Spin at 500g (1500RPM) for 7 minutes. 10- Remove the supernatant, agitate the cellular sediment and add drop-by- drop (with continous vortexing), 5ml of fresh, ice-cold fixative. 11- Leave at 4ºC for 20 minutes. 12- Repeat steps 9 and 10,until the supernatant is clear. 13- Spine at 500g (1500RPM) for 7 minutes. 14- Re-suspend the cell pellet with a 1.5ml of fresh fixative.

50 15- Drop 4-5 drops, from a high of approximately 30cm onto a clean slide and blow carefully on the drops for spreading them on the slide. 16- Put the slides on a 45ºC heated plate for 2-4 minutes. 17- Heat the slides to 60 ºC for overnight or to 90 ºC for 90 minutes. 18- Place the slides and flood them with Giemsa stain solution for 8 minutes. 19- Gently rinse the slides in distilled water and air dry. 20- Observe the chromsomes under microscope by using 10, 40 and 100x and photograph it and cut each chromosome from the photograph and arrange the chromosomes according to the size and position of centomer.

51 Size. Position of the centromere. Specific banding patterns.

52

53

54