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

OBJECTIVEREVIEW OF LITERATUREINTRODUCTIONMORPHOLOGYCLASSIFICATIONCLIMATIC CONDITIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSION

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.

Genetic variation and its importance

Sources of genetic variation

Genetic variation is also called as genetic diversity.

Genetic diversity is a necessary for survival in a world full of changing environmental stresses.

Genetic diversity provides the means for species to adopt to changing environmental conditions in future.

Mutation( changes in one or more letters of a DNA sequence)

Mutation come in the form of single base pair (point mutation), deletion, insertion, translocation or inverse of genes.

Recombination is the shuffling of DNA segments

Transgenic, a tool of modern biotechnology.

REVIEW OF LITERATURE

TECHNIQUES TO DETECT GENETIC VARIATION

TRADATIONAL: Studying the character tics

of plants & animals that are easy to observe & measure.

DNA BASED : Techniques to analyze

DNA, developed within the last 20 years, enabling to locate specific DNA chromosome.

GENE FLOW : A fair degree of genetic

modification occurs through gene flow during sexual reproduction, followed by natural selection.

CLONE/SEQUENCE BASED MARKER :

Single base pair polymorphic restriction site seq & measurable insertion or deletion

It can be detected by electrophoretic techniques

FINGER PRINT MARKER : Most commonly in use are the

minisatellites. Minisatellites is the

comparatively low upfront cost of detecting.

FUTURE BASED MARKER TECHNOLOGY :

The generation of large insert clone libraries for agriculture animal species is already underway.

INTRODUCTIONORIGIN: The orgin of chillies is believed

to be as old as 7000 B.C. In 1888, experiments began for

cross breeding of chili plant. It was originated from mexico. Later new breed of chilli plants

were evolved by crossbreeding. New variety of chilli i.e.

Anaheim was grown on 1906. There are more than 400

varieties of chilli grown all over the world

The hottest chilli is “Naga Jolokia” which is cultivated in hilly terrain of Assam.

DISTRIBUTION: Capsicum was first

introduced into Spain by Columbus in 1943.

Its cultivation spread from Mediterranean to England by 1948.

Chilli is actually reported to nature of south america.

Its cultivation was known to be native of Peru.

This crop was introduced to India by Portuguese towards the end of 15th century.

It became popular in 17th century.

MORPHOLOGY

BRANCHING: Chilli pant is highly

branched herbaceous plant. Its height ranges from 50 –

100cm Leaves are simple,

alternative with unequal margin.

FLOWER: Flower sometimes occur in

pairs. It is bell shaped, slender &

terminal.

FRUIT: Fruit is berry. Seeds are hot & embedded

on pericarp. Pericarp is leathery which

turns from green to purple, purple to red.

Fruit attain full maturity in around 35 days.

SEED: Seed start developing from

15 days of anthesis. Diameter of seed varies

from 3-4 mm.

CLASSIFICATION

Kingdom : PlantaeDivision : Magnoliophyta Class : MagnoliopsidaSubclass : AsteridaeOrder : SolanalesFamily : SolanaceaeGenus : CapsicumSpecies : frutescens

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.

MATERIALS AND METHODS

SAMPLE COLLECTION

SAMPLE COLLECTION

7 verities of chili plants were collected from I.I.H.R agri university, Bangalore. The samples are stored at 4⁰c.

Mahabharath Sarca arokaSamruthiindam 5f1 hybridIndam jwalaSBL-C

PLASMID ISOLATION

100μl of B. glycerol stock was added into 50ml of lb broth containing antibiotics

The conical flasks were incubated overnight at 37°C on the orbital shaker.

The cultures were transferred into sterile centrifuged tubes & chilled in an ice bath for 10mins at 4°C.

The cells were harvested by centrifuge at 6,000rpm for 6mins at 4°C.

Collect the pellet & then add 0.2ml of ice-cold solutin1 by using cyclo mixer.

Add 0.4ml of solution 2, mix it proper (RT)

Add 0.3ml of solution 3 and mix it gently & incubate in ice cold condition for 8-10mins.

The centrifugation at 12000rpm for 12 mins

collect supernatant and transfer to 2ml eppendorf tubes & 10-12μ was added

collect the upper layer & transfer to 2ml eppendorf tubes.

Add equal vol of chloroform:isoamyl alcohol (24:1)

vial was centrifuged at 10,000rpmfor 12mins

pellet was washed with ice cold 70% ethanol

the pellect was dried in speed vacuum desiccators.

ISOLATION OF GENOMIC DNA(CTAB METHOD)

DAY10.15g - 0.3g in 700- 900μl of CTAB buffer & crush with the help of Motor & pestle  Transfer to 2ml of vials & incubate T 50°C for 15mins in water

bath After incubation add equal vol of chloroform: isoamyl alcohol

(24:1) Incubate at 37°C for 30mins in shaker Centrifuge at 12000rpm for 12mins at R.T Collect the upper layer & transfer t 2ml vials. Add 0.5v 5M Nacl & mix it well & then add full vol of isopropanol Storage for -20°C overnight

DAY2Centrifuge at 12000rpm for 12mins

at 4°C

 

Collect pellet & allow to air dry for 10mins

 

After air dry add 800of TE buffer

 

Add 6 of Rnase & incubate at 37°C for 30mins in the bath

 

After incubation add equal vol of phenol: chloroform: isoamly alcohol (25:24:1)

 

Centrifuge at 12000rpm for 12mins at R.T

After centrifuge we get three layer

 

Collect the upper layer & transfer to 2ml vials

 

Add equal vol of chloroform : isoamyl alcohol & mix it gently

 

Centrifuge at 12000rpm for 12mins at R.T

 

Collect upper layer & transfer to 2ml vials

 

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

12000rpm for 12mins at 4°C

 Collect the pellet & then

add 1.5ml 70% ethanol Dissolve pellet gently Centrifuge at 12000rpm

for 12mins at 4°C

Collect pellet & air dry for 10 – 15 mins

 Add TE buffer  Dissolve the pellet

gently Prepare 0.8% of

agarose gel Load the 10μl genomic

DNA

SPECTROPHOTOMETRIC QUANTIFICATION OF DNA

REQUIREMENTS :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 PCR

REQUIREMENTS :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

S.No

DNA sample volume(µl)

Positive control (µl)

dNTPs (µl) Buffer (10x)(µl)

Forward primer (µl)

Reverse primer (µl)

Taq polymerase (µl)

Sterilewater (µl)

1 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0

2 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0

3 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0

4 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0

5 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0

6 8.0 3.0 2.25 2.5 0.75 0.75 0.75 7.0

Reaction components:

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 30Segment 94ºC 30secSegment - 55ºC - 30secSegment - 72ºC - 1minute

Final extention 72ºC - 5minutesFinal hold 10ºC

AMPLIFICATION OF DNA USING RAPD

REQUIREMENTS : 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

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

PROCEDURE:Set up the following reaction mixture (25 l) in the same order.

All those mentioned ingredients are mixed and prepared for the total no of reactns including a blank with a particular primer excluding template The calculated volume of masters mix ix then transferred to labeled PCR tubes with template source and primer. Finally 0.33µl of Taq DNA polymerase is added to each tube The contents of the tube are mixed with a brief spin and transferred to PTC 200 thermal cycler 

The program with following conditions is selected for the amplification Number cycles 30Segment 94.0ºC 1minuteSegment 35.0ºC 1minuteSegment 72.0ºC 1minute

UREA POLYACRYLAMIDE GEL ELECTROPHORESIS

REQUIREMENTS :Vertical electrophoresis unitUrea 7MAcrylamide 40%10x TBE (Tris Borate EDTA) buffer10%Ammonium Per Sulphate (APS)Tetra Ethyl Methylene Diamine (TEMED)Gel loading dyeAutoclaved 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

SILVER STAINING

REQUIREMENTS :Gel containerShaker incubator10% acetic acidde-ionised waterautoclaved double distilled watersilver nitrate solution2.5% sodium carbonate and 0.02%

formaldehyde.

PROCEDURE:Place the gel in 5 volumes of a mixture of 30% ethanol and 10% acetic acid.

 Incubate the gel for 3 hours or

overnight with shaking at room temperatures.

 Remove the ethanol / acetic

acid solution and add 5 gel volume of 30% ethanol.

 Incubate for 30minutes at room

temperatures with shaking. Repeat this step twice.

 Remove the ethanol solution

and add 10 gel volume of deinonised water.

Incubate for 10minutes at room temperatures with shaking. Repeat this step twice.

 Remove the deionised water

and add 5 gel volume of 0.1%silver nitrate solution.

 Incubate for 30minutes at

room temperatures with shaking.

 Remove the silver nitrate

solution and wash the gel for 20seconds under a stream of deionised water.

 Add 5 gel volume of a mixture

of 2.5%sodium carbonate and 0.02% formaldehyde

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 each

The 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.

RESULTS AND DISCUSSION

GENOMIC DNA ISOLATION

In the present study DNA was isolated from chilli leaves following the CTAB method.

Method described by Doyle (1987) with few modification.

About 320 g of pure G.DNA could be isolated by this method.

AGAROSE GEL ELECTROPHORESIS

After isolation of G.DNA from chilli leaves sample were loaded into 0.8% agarose gel.

To cross check the presence or absence of G.DNA in isolated sample.

In agarose gel we observed 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 PCRThe 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.

1

2

3

4

5

6

7

8

9

10

CTATAAGCCA

GGTGACGCAG

CCGGTGTGGG

TGCCCGTCGT

CCCTGTCGCA

TAGCCTAGGC

CTGAGACGGA

GGCAGCAGGT

GAATGCGACG

ATGACGTTGA

Primers used and their sequence in 5’ to 3’ direction

UREA ACRLY AMIDE GELWell 1 – samruthi, primer 4Well 2 - mahabharath,

primer 3Well 3-samruthi, primer 2Well 4- f1 hybrid, primer 2Well 5- mahabharath,

primer 2Well 6- indam 5, primer 1Well 7- ladder dnaWell 8- mahabharath,

primer 4Well 9- samruthi, primer 4Well 10- f1 hybrid, primer 4Well 11- jwala, primer 3Well 12- jwala, primer 4Well 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.

THANK YOU