practical of genetics
DESCRIPTION
Practical Of Genetics. Lab 4 & 5 Human Karyotype. Objectives:. 1. Students will be able to demonstrate a microtechnique for reliable chromosomal analysis of leucocytes obtained from peripheral blood. - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/1.jpg)
![Page 2: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/2.jpg)
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.
Objectives:
![Page 3: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/3.jpg)
![Page 4: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/4.jpg)
![Page 5: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/5.jpg)
INTRODUCTION
![Page 6: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/6.jpg)
INTRODUCTION
![Page 7: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/7.jpg)
Triploidy
![Page 8: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/8.jpg)
![Page 9: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/9.jpg)
![Page 10: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/10.jpg)
![Page 11: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/11.jpg)
![Page 12: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/12.jpg)
![Page 13: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/13.jpg)
![Page 14: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/14.jpg)
![Page 15: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/15.jpg)
![Page 16: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/16.jpg)
XYY Syndrome: 47, XYY
![Page 17: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/17.jpg)
Detecting Cancer
![Page 18: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/18.jpg)
![Page 19: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/19.jpg)
![Page 20: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/20.jpg)
• A chromosome is divided by its centromer into short arm p (= petite or short) and long arm q (= queue, or long)
p
q
![Page 21: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/21.jpg)
Chromosomes can be classified by the position of their centromer:
![Page 22: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/22.jpg)
![Page 23: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/23.jpg)
• Humans do not possess any telocentric chromosomes.
![Page 24: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/24.jpg)
![Page 25: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/25.jpg)
Chromosomes are arranged into seven groups
AA 1-3 Large metacentric
BB 4,5 Large submetacentric
CC 6-12, X Medium submetacentric
DD 13-15 medium acrocentricDD 13-15 medium acrocentric
EE 16-18 short metacentric
FF 19-20 Short metacentric
![Page 26: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/26.jpg)
![Page 27: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/27.jpg)
![Page 28: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/28.jpg)
![Page 29: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/29.jpg)
![Page 30: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/30.jpg)
• Samples for chromosomal analysis can be prepared relatively easily using skin, bone morrow, chorionic villy or cells from amniotic fluid.
![Page 31: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/31.jpg)
![Page 32: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/32.jpg)
![Page 33: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/33.jpg)
• 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.
![Page 34: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/34.jpg)
![Page 35: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/35.jpg)
![Page 36: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/36.jpg)
![Page 37: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/37.jpg)
– Heparinzed whole blood– Heparin sodium injection– Peripheral blood Karyotyping medium with PHA (RPMI
1640)– Incubator 5% CO2 at 37 °C
– Colcemide solution 10g/ml– 0.075 M KCl– Fixative solution ( 3x methanol : 1x glacial acetic acid )– Giemsa stain solution– Slides and Microscope
Metirals
![Page 38: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/38.jpg)
Peripheral blood media preparation
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 4C for along as 2 weeks.
![Page 39: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/39.jpg)
Stains and Dyes
• 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
![Page 40: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/40.jpg)
1- Inoculate 0.5ml of heparinized whole blood into tube with 10ml of
karyotyping medium. 2- Incubate the tubes in incubator with 5% CO2 at 37 oC 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 oC for an additional 20-30 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 37oC.
Karyotyping procedure
![Page 41: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/41.jpg)
7- Incubate at 37oC 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.
![Page 42: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/42.jpg)
15- Drop 4-5 drops, from ahigh of approximately 30cm onto aclean 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.
![Page 43: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/43.jpg)
Staining procedure
Materials:1. Giemsa stain solution - 6ml Giemsa stain in
70ml ddH202. HBSS3. Trypsin X104. PBS without Ca, Mg
![Page 44: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/44.jpg)
Method
1. Incubate the slides in Trypsin solution:2ml Trypsin x10 in 50ml of HBSS at room temperature.
2. After 2.5 min., neutralize the trypsin by immersing the slides in PBS with 5% FCS.
3. Rinse the slides in PBS.4. Place the slides horizontally and flood them with stain
solution for 2.5 minutes.5. Gently rinse the slides in ddH20 and air-dry.• Exact times of Trypsin treatment and staining should be
determined personally by each lab to get optimal results
![Page 45: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/45.jpg)
Chromosomes are arranged by :
• Size.• Position of the centromere.• Specific banding patterns.
![Page 46: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/46.jpg)
![Page 47: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/47.jpg)
![Page 48: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/48.jpg)
![Page 49: Practical Of Genetics](https://reader035.vdocuments.net/reader035/viewer/2022062519/56815570550346895dc33e2f/html5/thumbnails/49.jpg)