WT1 GENE MUTATION project

8/7/2019 WT1 GENE MUTATION project

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Indexy Introduction

y Aim of the project

y Materials & reagents

y Methodology

y

Remaining worky Expected result

y Conclusion


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Introduction

y W

T1 gene:- wilms Tumour gene* This gene encodes a transcription factor that contains four

zinc-finger motifs at the C-terminus and aproline/glutamine-rich DNA-binding domain at the N-

terminus. It has an essential role in the normal developmentof the urogenital system.

present on the chromosome no.-11p13

Description10 exons spanning 48 kb of genomic DNA.

Transcription3 kb mRNA; four alternative splice forms: +/-exon 5 and alternative splice donor sites at exon 9.


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Nephrotic syndrome

y

Nephrotic syndrome is a nonspecific disorder in whichthe kidneys are damaged, causing them to leak largeamounts of protein (proteinuria )from the blood intothe urine.

y

Kidneys affected by nephrotic syndrome have small pores inthe podocytes, large enough to permit proteinuria andsubsequently hypoalbuminemia,hyperlipidemia,edema.


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View of projecty Aim of the project

y WT1 gene mutation a causefor Nephrotic syndrome.

y

Type of mutation &response to the treatment.

y Importance of project

y In Indianpopulation,mutationalanalysis inWT1 gene onnephrotic syndrome hasnot been performed inpatients.

y Nature & type of mutation& its association with thenephrotic syndrome isunclear in Indianpopulation.


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Apparatus & Reagentsy Apparatusy

Eppendroffy MicroPippetey Centrifugey Gel electrophoretic tanky PCRy Seqencer 3730 modely

Laminar air flowy Vortex machine

y ReagentsDNA extraction(KIT method)

-red cell lysing buffer(RCLB)-tritonX-nucleated lysing buffer(NLB)-sodium dodecyl sulphate(SDS),Nacl-70% ethanol-RNAse free waterGel electrophoresis-agarose-1x TAE buffer-EtBr(ethidium bromide)-bromophenol bluePCR-mastermix

-forward & reverse primer-buffer,water-template DNASeqenncing-ready reaction mix-dilution buffer,water-template DNA

-primer


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METHODOLOGY

Blood sample from the 10 different patients are collected,3ml of blood from each samplehas been taken in centrifuge tube.Add RCLB.make upto 10ml.

Add Triton X(100l).incubate at 37C for 5mins,spin at 2000 rpm for 15 mins.Discard the supernatant and add RCLB(10 ml).Vortex and mixing.spin at 2000 rpm for 15

mins(step is repeated until it turns white pellete).add 1 ml NLB and 20l SDS.incubate inwater bath at 55C for 1 hr.

Transfer the whole sample into the eppendroff tube (2ml).add 400l Nacl.spin at10,000rpm for 10-15 mins.take the supernatant and add to 15 ml tube.Add absolute Alcohol

double the volume of supernatant.

Scoop the DNA,put it in a eppendroff and add 500l of 70% Ethanol.spin at10,000rpm.Discard the supernatant .Dry the pellet for 2-3 hrs.Add 100l of RNAse free

water.

DNA EXTRACTION


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0.2 gm of Agarose in 25ml 1x TAE buffer.boil for 2min.Add 4l of EtBr .Cast the gel

Add Bromophenol blue(1l) & 10l of DNA. Mix well & load the molecular ladder by usingmicropipette. Run the gel at 50 /100 volts for 30/15 mins.Observe the result in UV

transilluminator.

Take micro centrifuge tube.& take 6l of mastermix,add 0.2l of forward & reverseprimer.add 1.6l of nuclease free water. At the last add 2l of template DNA,in each tube.

Set the PCR programe in PCR mechine prior to use.keep the centrifuge tube in PCRmachine & run for the 30 cycles.

Perform the gel electrophoresis at 150 volts for 15min,for checking the purity of the PCR

amplification.observe the result in UV transilluminator.

GEL electrophoresis

PCR

Check for the PCR amplification


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Remaining work & expected result

Remaining works

Mutational analysis of the DNA sample for WT1 gene.

Expected result

Nature & type of mutation in WT1 gene.WT1 gene mutation & response to the treatment.

Mutation frequency

Results obtained

DNA has been extracted successfully from the nephrotic syndrome patients.Quality & quantity of the DNA has been checked.it reveals a good quality DNA.

PCR work is done and amplified DNA has been obtained.


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WT1 GENE MUTATION project

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