Identification of polymorphism by dna fingerprinting using rapd 2
IDENTIFICATION OF POLYMORPHISM BY DNA FINGERPRINTING USING RAPD IN CHILLI Under the Guidance Dr. Deepak.R (HOD) External Guidance Govind Rao Submitted by Abhinav Varma (1CR08BT001) Sachin Subba (1CR09BT400) Syed Mubasir (1CR05BT026) Department of Biotechnology CMRIT Bangalore.
CONTENTS OBJECTIVE REVIEW OF LITERATURE INTRODUCTION MORPHOLOGY CLASSIFICATION CLIMATIC CONDITION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSION
OBJECTIVE The objective of this project is to examine the technology of DNA fingerprinting. Identification of polymorphism by DNA fingerprinting using RAPD in different variety of chilli considered for the present study. 7 varieties of chillies were selected for the study. This work was carried out under the SBL bangalore. Isolation of genomic DNA. Agarose gel electrophoresis of PCR product. Spectrophotometric quantification. Techniques to detect genetic variation of DNA.
REVIEW OF LITERATURE Sources of genetic variationGenetic variation and itsimportance Genetic variation is also Mutation( changes in one called as genetic or more letters of a DNA diversity. sequence) Genetic diversity is a Mutation come in the form necessary for survival in of single base pair (point mutation), deletion, a world full of changing insertion, translocation or environmental stresses. inverse of genes. Genetic diversity Recombination is the provides the means for shuffling of DNA segments species to adopt to Transgenic, a tool of changing environmental modern biotechnology. conditions in future.
TECHNIQUES TO DETECT GENETIC VARIATIONTRADATIONAL: CLONE/SEQUENCE BASED Studying the character tics of MARKER : plants & animals that are easy Single base pair polymorphic to observe & measure. restriction site seq & measurable insertion or deletionDNA BASED : It can be detected by Techniques to analyze DNA, electrophoretic techniques developed within the last 20 FINGER PRINT MARKER : years, enabling to locate Most commonly in use are the specific DNA chromosome. minisatellites. Minisatellites is theGENE FLOW : comparatively low upfront cost A fair degree of genetic of detecting. modification occurs through FUTURE BASED MARKER TECHNOLOGY : gene flow during sexual The generation of large insert reproduction, followed by clone libraries for agriculture natural selection. animal species is already underway.
INTRODUCTIONORIGIN: DISTRIBUTION: The orgin of chillies is believed to Capsicum was first introduced be as old as 7000 B.C. into Spain by Columbus in 1943. In 1888, experiments began for Its cultivation spread from cross breeding of chili plant. Mediterranean to England by 1948. It was originated from mexico. Chilli is actually reported to Later new breed of chilli plants nature of south america. were evolved by crossbreeding. Its cultivation was known to be New variety of chilli i.e. Anaheim native of Peru. was grown on 1906. This crop was introduced to There are more than 400 India by Portuguese towards the varieties of chilli grown all over end of 15th century. the world It became popular in 17th century. The hottest chilli is “Naga Jolokia” which is cultivated in hilly terrain of Assam.
MORPHOLOGYBRANCHING: FRUIT: Chilli pant is highly branched Fruit is berry. herbaceous plant. Seeds are hot & embedded on Its height ranges from 50 – pericarp. 100cm Pericarp is leathery which Leaves are simple, alternative turns from green to purple, with unequal margin. purple to red. Fruit attain full maturity inFLOWER: around 35 days. Flower sometimes occur in pairs. SEED: It is bell shaped, slender & Seed start developing from 15 terminal. days of anthesis. Diameter of seed varies from 3-4 mm.
CLIMATIC CONDITON It can be grown in both warm & cold climatic condition. The ideal temperature range is 20-25°C. The crop is killed in freezing temp and frost. Heavy continues rain during flowering results in poor fruit. High temp. & dry winds are injurious to plant.
SAMPLE COLLECTIONSAMPLE COLLECTION7 verities of chili plants were collected from I.I.H.R agri university, Bangalore. The samples are stored at 4⁰c. Mahabharath Sarca aroka Samruthi indam 5 f1 hybrid Indam jwala SBL-C
PLASMID ISOLATION Add 0.3ml of solution 3 and mix it gently & incubate in ice cold condition for 8-10mins. 100μl of B. glycerol stock was added into 50ml of lb broth containing antibiotics The centrifugation at 12000rpm for 12 mins The conical flasks were incubated overnight at 37 C on the orbital shaker. collect supernatant and transfer to 2ml eppendorf tubes & 10-12μ was added The cultures were transferred into sterile centrifuged tubes & chilled in an ice bath collect the upper layer & transfer for 10mins at 4 C. to 2ml eppendorf tubes. Add equal vol of The cells were harvested by centrifuge at chloroform:isoamyl alcohol (24:1) 6,000rpm for 6mins at 4 C. vial was centrifuged at 10,000rpmfor 12mins Collect the pellet & then add 0.2ml of ice- cold solutin1 by using cyclo mixer. pellet was washed with ice cold 70% ethanol Add 0.4ml of solution 2, mix it proper (RT) the pellect was dried in speed vacuum desiccators.
DAY10.15g - 0.3g in 700- 900μl of CTAB buffer& crush with the help of Motor & pestleTransfer to 2ml of vials & incubate T 50°C for 15mins in water bathAfter incubation add equal vol of chloroform: isoamyl alcohol (24:1)Incubate at 37°C for 30mins in shakerCentrifuge at 12000rpm for 12mins at R.TCollect the upper layer & transfer t 2ml vials.Add 0.5v 5M Nacl & mix it well & then add full vol of isopropanolStorage for -20°C overnight
DAY2Centrifuge at 12000rpm for 12mins at 4 C After centrifuge we get three layerCollect pellet & allow to air dry for Collect the upper layer & transfer to 2ml 10mins vialsAfter air dry add 800of TE buffer Add equal vol of chloroform : isoamyl alcohol & mix it gentlyAdd 6 of Rnase & incubate at 37 C for 30mins in the bath Centrifuge at 12000rpm for 12mins at R.TAfter incubation add equal vol of phenol: chloroform: isoamly alcohol (25:24:1) Collect upper layer & transfer to 2ml vialsCentrifuge at 12000rpm for 12mins at R.T Add 0.1v 3m sodium acetate & mix it well Add full vol of absolute ethanol & mix it well Store at -20 C overnight
DAY3Mix & centrifuge at Collect pellet & air dry for 12000rpm for 12mins at 10 – 15 mins 4°C Add TE bufferCollect the pellet & then add 1.5ml 70% ethanol Dissolve the pellet gentlyDissolve pellet gently Prepare 0.8% of agarose gelCentrifuge at 12000rpm for 12mins at 4°C Load the 10μl genomic DNA
SPECTROPHOTOMETRIC QUANTIFICATION OF DNAREQUIREMENTS : UV spectrophotometer TE buffer DNA sample Micropipette Absolute Ethanol
PROCEDURE:Prepare a known dilution of DNA sample in the TE buffer, which is used to dissolve the DNA sample.Calibrate the spectrophotometer for blank using TE buffer.Record the OD of the sample at 260nm and 280nm.Calculate the concentration of DNA in the sample using the Relation
QUALITY PCRREQUIREMENTS : Thermo stable Taq DNA polymerase dNTP mix (10mM) Chili genomic DNA Sterile distilled water PCR buffer (10x) Forward primers and reverse primer specific to positive control Micropipettes of different ranges
QUALITY PCR PROGRAMHeated lid 110ºC Pre- heated lid off Pause- off Initial denaturation- offLoop 1 (initial denaturation) No. of cycles 1 Segment 94ºC 3minutesLoop 2 No. of cycles 30 Segment 94ºC 30sec Segment - 55ºC - 30sec Segment - 72ºC - 1minuteFinal extention 72ºC - 5minutesFinal hold 10ºC
AMPLIFICATION OF DNA USING RAPDREQUIREMENTS : Thermostable Taq DNA polymerase dNTP mix (10 mM) Template DNA Sterile distilled water PCR buffer (10x) Oligonucleotide primers Ice bucket Eppendorff vials Micropipettes of different ranges Thermal cycle
PROCEDURE:Set up the following reaction mixture (25 l) in the same order. Ingredients Volume to be taken Template DNA 10.0μl dNTPs 2.5μl PCR buffer 2.5μl Primers 1.0μl Taq DNA polymerase 0.75μl Sterile water 8.25μl Total 25μl
All those mentioned ingredients are mixed and prepared for thetotal no of reactns including a blank with a particular primerexcluding templateThe calculated volume of masters mix ix then transferred tolabeled PCR tubes with template source and primer.Finally 0.33µl of Taq DNA polymerase is added to each tubeThe contents of the tube are mixed with a brief spin andtransferred to PTC 200 thermal cyclerThe program with following conditions is selected for theamplificationNumber cycles 30Segment 94.0ºC 1minuteSegment 35.0ºC 1minuteSegment 72.0ºC 1minute
UREA POLYACRYLAMIDE GEL ELECTROPHORESISREQUIREMENTS : Vertical electrophoresis unit Urea 7M Acrylamide 40% 10x TBE (Tris Borate EDTA) buffer 10%Ammonium Per Sulphate (APS) Tetra Ethyl Methylene Diamine (TEMED) Gel loading dye Autoclaved distilled water
PROCEDURE:Preparation of gel (50ml) Weigh 9.08g of urea and dissolved by heating in about 15ml autoclaved distilled water. Add 6.25ml of 40% acrylamide and 5ml of 10x TBE buffer. Make up the volume to 50ml with autoclaved distilled water. Add 350μl of APS and 35l of TEMED and mix well. Immediately transfer the gel into the previously arranged vertical electrophoresis unit.
Electrophoresis of the DNAPre-run the gel for about one hour at 100V.To the PCR sample add 4.2l of gel loading dye.(Xylene Cyanol).Boil the samples for 10minutes at 85-90C.Immediately chill the sample in ice for 2minutes.Spin the sample at 3000rpm for 2minutes and load in top the gel.The electrophoresis is carried out at 150V tll the dye front reaches the bottom of the base plate, the plates are cooled with an ice pack during the run to prevent over-heating
PROCEDURE: Incubate for 10minutes at roomPlace the gel in 5 volumes of a temperatures with shaking. mixture of 30% ethanol and Repeat this step twice. 10% acetic acid. Remove the deionised water andIncubate the gel for 3 hours or add 5 gel volume of 0.1%silver overnight with shaking at room nitrate solution. temperatures. Incubate for 30minutes at roomRemove the ethanol / acetic acid temperatures with shaking. solution and add 5 gel volume of 30% ethanol. Remove the silver nitrate solution and wash the gel forIncubate for 30minutes at room 20seconds under a stream of temperatures with shaking. deionised water. Repeat this step twice. Add 5 gel volume of a mixture ofRemove the ethanol solution and 2.5%sodium carbonate and add 10 gel volume of 0.02% formaldehyde deinonised water.
Incubate at room temperature with shaking. Bands will start appearing slowly.Incubate until band appears.Stop the reaction by washing with 1% acetic acid.Wash several times with deionised water for 10 minutes eachThe gel might now be observed over an illuminating source of white light for better result and documented.For preserving the gel, place it in 20ml of a 20% glycerol solution.Keep the gel between two layers of gelatin [aper and dry for 3 days at 37ºC.
GENOMIC DNA ISOLATION AGAROSE GEL In the present study DNA ELECTROPHORESIS was isolated from chilli After isolation of G.DNA leaves following the CTAB from chilli leaves sample method. were loaded into 0.8% Method described by Doyle agarose gel. (1987) with few To cross check the presence modification. or absence of G.DNA in About 320 g of pure G.DNA isolated sample. could be isolated by this In agarose gel we observed method. respective G.DNA bands with little streaking. These streaking might be because breakage of DNA.
QUALITY PCR To cross check quality of G.DNA in test sample We arrange quality PCR using test chilli G.DNA sample. +ve control along with specific primer, loaded into 0.8% agarose gel. Out of 7 chilli varities only 6 varities were amplified throughout along with +ve control Indicates only 6 chilli G.DNA quality was good.
RAPD PCR The RAPD technique was standardize by adapting various temp, primer conc. After standardize of RAPD program routine analysis was done with a PCR. Program having 2mins initial denauration (94⁰c) . 1min denauration (94⁰C) 1min annealing (35⁰c) 1min extension (72⁰c) For about 30 cycles, this was followed by one final extension 72⁰c for about 5mins.
SCREENING OF RAPD PRIMERS RAPD analysis of isolated chilli plant G.DNA was carried out with 10 different oligonucleotide random primer. Out of 10 random primers tested for chilli only 4 primers showing amplification with test sample. So that we use only 4 selected primers to study polymorphism in 6 chilli varities.
PRIMER 1 Produce moderate level of polymorphic in 6 chilli verities. F1 hybrid showing 4 amplified fragments with different molecular weight. Samruthi, indam5 & jwala showing 3 amplified fragments with different mol.wgt Magabharathi showing 2 amplified fragments with different mol.wgt
PRIMER 2 Produce moderate level of polymorphic fragments in 6 chilli verities. F1 hybrid, magabharathi showing 2 amplified fragments with different mol.wgt Samruthi showing 1 amplified fragments. Indam 5 & aroka suphar no fragments.
PRIMER 3 Produces moderate level of polymorphic bands in 6 chilli verities. F1 hybrid showing 4 amplified fragments with different mol.wgt Magabharathi, indam5, samruthi, jwala showing 3 amplified fragments. Aroka suphar showing 2 amplified fragments.
PRIMER 4 Produce moderate level of polymorphic fragments in 6 chilli verities as depicted in fig. Samruthi showing 4 fragments Indam 5 showing 3 fragments Magabharathi showing 2 fragments. Jwala no fragments.
Primers used and their sequence in 5’ to 3’ direction1 CTATAAGCCA2 GGTGACGCAG3 CCGGTGTGGG4 TGCCCGTCGT5 CCCTGTCGCA6 TAGCCTAGGC7 CTGAGACGGA8 GGCAGCAGGT9 GAATGCGACG10 ATGACGTTGA
UREA ACRLY AMIDE GEL Well 1 – samruthi, primer 4 Well 2 - mahabharath, primer 3 Well 3-samruthi, primer 2 Well 4- f1 hybrid, primer 2 Well 5- mahabharath, primer 2 Well 6- indam 5, primer 1 Well 7- ladder dna Well 8- mahabharath, primer 4 Well 9- samruthi, primer 4 Well 10- f1 hybrid, primer 4 Well 11- jwala, primer 3 Well 12- jwala, primer 4 Well 13- mahabharath, primer 3
CONCLUSION Polymorphism between genotypes is due to either a nucleotide base change that alters the ability of the primer to anneal to the DNA template within the amplified fragment. All the primers cannot amplify all the verities of chilies. Variation was seen as certain primers could separate. Our study reflected the tremendous genetic diversity available among the genotypes. The rich genetic diversity in which breeding efforts depend can be utilized for current & future.