Restriction Digestion 1

RESTRICTION DIGESTION OF HUMAN METAPHASE CHROMOSOMES By Anita Swaminathan Siddharth krishnan Suchithra Seshadrinathan G.Vimal Guide : Dr.S.Meignanlakshmi

Objectives • Harvesting and culturing of Human Peripheral Blood. • Identify the most effective method to maximize growth of lymphocytes • Standardization of Karyotyping protocol to the present lab conditions • Obtain spreads of Human Metaphase Chromosomes • Restriction Digestion of Human Metaphase Chromosomes

Karyotyping • Karyotyping is a diagnostic tool that allows us to check the images of the structure and the number of chromosomes in an individual’s somatic cell. The preparation of an individual’s Metaphase chromosomes sorted out by their distinctive visual features is called a Karyotype. • Any abnormalities in the chromosome number or structure can be identified by comparing a standard karyotype for that species. • Obtaining results depends on various external factors hence a protocol has to be standardized for specific lab conditions

Different methods tested for obtaining the effective culture • • • •

Whole Blood culture method Whole Blood Centrifugation Ficol-Paque Centrifugation Method RBC lysis Buffer Method

Harvesting and culturing of Human Peripheral Blood. • 5 ml of Whole blood was drawn and added to a tube containing 40 microliters of Heparin. • 2.5 ml of Heparinized Blood was added to autoclaved Centrifuge tube containing 7 ml of RPMI1640 medium and 3 ml of filtered Bovine calf serum • The tube is incubated in a normal Incubator at 37 deg C for 72 hours.

Whole Blood Centrifugation Method • 5 ml of Whole blood was drawn and added to a tube containing 40 microliters of Heparin. • 2.5 ml of Heparinized Blood was centrifuged at 2000 rpm for 15 minutes. • Supernatant is innoculated into 7ml of Medium with 3ml of Serum and incubated at 37deg C for 72 hours.

Ficol-Paque Centrifugation Method • 5 ml of Whole blood was drawn and added to a tube containing 40 microliters of Heparin. • 1.5ml of Balanced Salt Solution is added to 2.5 ml of Heparinized Blood. • This mixture is added to 1.5ml of Ficol-Paque and centrifuged at 2000 rpm for 15min • White layer obtained which is rich in lymphocytes is added to the fresh medium • Incubation : 72hrs at 37 deg C

RBC lysis buffer method

The follow-up procedure • Culture was treated with 0.075M KCl for varying time periods and centrifuged. • Treated with Fixative (different fixatives were tried) and centrifuged several times. • cell solution is dropped using a pipette from a minimum of 2ft height at angle of 45 deg on to a slide for bursting of cells and cause spreading of chromosomes. • Slides are treated with Giemsa stain and viewed under microscope.

Results obtained .. • Whole blood culture yielded the most satisfactory spreads, but improvements are to be made to reduce the amount of RBC’s so that better spreads could be obtained. • After several trials It was found that Kcl treatment incubation for 1 hour was the optimum incubation period to cause better spreads • Geimsa staining for 1 hour produces better stains. • Different fixatives were tried and it was found that methanol-acetic acid fixative was the most effective.

Work plan • To try to obtain perfect chromosome spreads • To subject these spreads to restriction digestion by Msp1 and Alu1