He Vol.

1(2):152-154 (201 elix 12)

Isolatio and Q on Quantification of DN from different evidence and rol NA t es le of RF FLP in DN finger NA rprinting
Ninad Lax xman Harkulk * kar
Tyatasahe Kore Institute of Engineering and Technology eb y
Phone: 08087564557, Ema ninadharkulk 0 ailkar@gmail.com

Receive January 27, 2 ed 2012; Accepted F February 25, 201 Published M 12; March 01, 2012

Ab bstract: Ov the past tw ver wenty years, D DNA analysis has been revolutionalized forensic scie d ence and has become a b do ominant tool in law enfor rcement. The DNA is un nique for each and every per rson. This appl lication is us in forensic science to iden sed ntify the crimin nals. Th DNA is is he solated from the biological samples l co ollected from th crime scene DNA can be extracted he e. e fro saliva, blo om ood, hairs, sem or any bo fluid. men ody Th various mat he terials like cig garette, chewin gum or ng an material con ny ntaining body f fluids may con ntain DNA as it is used b the person. The person is then s by n id dentified by va arious methods like STR, R RFLP and VN NTR. The esti imation of the DNA is done by DPA e e method. m In ntroduction : DN NA forensic is the bed rock tool f for crime in nvestigation. It is one of t t the very accu urate and reliable measure that have bec es come the main source in n he ment. The DN is being fun NA ndamental th law enforcem str ructure of th life and a he always unique for the e in ndividual. 99.99% of the hu uman genome is similar an contains ex nd xons and intro ons. Only 0.1% of the DN is differe and gives the specificity to each NA ent y pe erson. During any case investigation various g n ph hysical, testim monial and biological k kinds of ev vidences may be found at th crime scen Out of he ne. th hese biological evidences are very importa These e ant. in nclude saliva, b blood, semen or any biological fluid. Th hese fluids co ontains variable amount of D e DNA and ca ensure the CSIOs that the source person of DNA an C e n wa present at the crime scen However it doesnt as ne. co onfirm the pe erson as the culprit or h direct his in nvolvement in the crime, b he cant r but refuse his pr resence on the crime scene. DNA forensic involves c co ollection of samples with proper c care and fin ngerprinting of DNA. The various techniqu in the f ues DN forensic in NA nclude PCR, STR, VNTR and RFLP S a etc Restriction Enzyme Len c. ngth polymorp phism and Sh Tandem R hort Repeats gives the band patte on the ern ge which is ve specific. U el ery Using these tec chnologies th individual ca be identifie The DNA forensic is he an ed. f also used in the paternity identification and the t missing cases. The qualificatio of DNA can be done m T on n by the Agarose gel electropho y oresis method. DNA can be estimated by DPA method. e Ke words: DNA Extraction of DNA, evidence, RFLP ey A,

Materials and Methods s s: Isolation of DNA: Sa amples were dipped in lys sis buffer and incubated f 1 hour. 50 l NaCl w for 00 was hen tes added. Th centrifuged at 4000rpm for 10 minut and treate with ice col isopropanol. Incubated at 40 ed ld . C for overnight. Per o rformed etha anol wash an nd centrifuge centrifuged at 10000 rpm for 10 minute ed es. DNA was stored in TE b s buffer. fer Running on gel: using 0.8 % agrose and TAE buff as nd he the gel wa prepared an the samples were run on th gel. DNA was observed under UV tran A nsilluminator. cation of DN NA: the vials were kept in s Quantific boiling wa bath and 4 of DPA rea ater 4ml agent was adde ed with 10 l of samples and water. T O.D. of th The he samples is measured at the 595nm and graph is m Using the gr raph, the con of DNA is nc. plotted. U estimated. RFLP: tw enzymes Ec and Hind III were used f wo corI I for RFLP. 7 of samples and 2 l of en l nzymes 2.5 l of assay buf were added and volume was made 25 l ffer d by water. The band patt terns were observed under U UV minator. transillum Results: alitative analy of DNA by gel ysis y Fig1: Qua electroph horesis:-

Fig 1: sho owing DNA ba ands on gel elec ctrophoresis 1st band = DNA from cig garette butt 2nd band = DNA from ch hewing gum 3rd band = DNA from lip pstick mark

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 Helix Vo 1(2):152-1 (2012) ol. 154

Es stimation of D DNA by DPA method: m Th deoxyribos of DNA in presence of acid he se dihydroxylevulin nal-dehyde which reacts with c diphenylamine to give a blue colour. tandard soluti ion: 1mg/ml of Calf thymus DNA in o s St distilled water. Ta able1: showing the Estima ation of DNA by DPA A method m ig2: The Grap showing the Concentration of ph e Fi DN in the unk NA known sample es
Vial No. Sample lume of vol DN (l) NA A DPA (ml) Dist. H 2O (l) Total volume (ml) O.D O va alues (595nm)

1. 2. 3.

Cigar rette butt U1= 125g g 10 l l 12 g 25 12.5 g/ l, 1.11mg g/ml g Lipstick mark U2= 110 10 l l 110 g 11 g l, 0.11mg/m g/ ml Chew wing gum g U3= 220 10 l l 220 g 0 22 g l, 0.22mg/m g/ ml

RFLP:-

S1 S2 S3 S4 S5 U1 U2 U3 B

100 200 300 400 500 10 10 10 --

3 3 3 3 3 3 3 3 3

0.9 0.8 0.7 0.6 0.5 ---1ml

4 4 4 4 4 3.01 3.01 3.01 4

0.07 7 0.20 0 0.45 5 0.60 0 0.75 5 0.19 9 7 0.17 0.33 3 0.0

Fig 2: sho owing different band pattern on Agarose ge t el after RFL LP The differ rent band patte on the gel was obtained as ern the sampl were collec les cted from different sources an nd not from the same indivi t idual. on: Conclusio DNA fore ensics techniqu is highly au ue uthentic and ve ery much acc curate. Each person has similarities an nd difference in DNA seq es quences. DNA forensic work A ks on the basic like extraction, isolation an nd tion. The sour person of the DNA can b rce t be identificat identified by using RFLP and STR. n rom the variou materials lik us ke DNA can be isolated fr cigarette, lipstick print or chewing gum used by th he person. Th quantity of t DNA howe the he ever differs. By above studies and r e results, we can conclude tha n at, the eviden nces concern w direct con with ntact with saliv va like chew wing gum are most importa as they ca ant an provide considerable am mount of DNA Such sampl A. les h y d. should be collected with prime priority and preserved

Fr rom the Grap the followin information can be ph ng ob btained: oncentration o DNA in the samples calc of e culated by Co gr raph:

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 Helix Vol. 1(2):152-154 (2012) References: 11. Recovery of DNA for Forensic Analysis from Lip Cosmetics*
1. Autosomal Short Tandem Repeat Analysis of Ancient DNA by Coupled Use of Mini- and Conventional STR Kits* Oh CS, Lee SJ, Park JB, Lee SD, Seo SB, Kim HY, Kim J, Kim YS, Shin DH. 12. REFERENCE: Webb LG, Egan SE, Turbett GR. Recovery of DNA for forensic analysis from lip cosmetics. J Forensic Sci 2001;46(6):14741479.

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Performance of two 17 locus forensic identification STR kits-Applied Biosystems's AmpFSTR() NGMSElect and Promega's PowerPlex() ESI17 kits. Tvedebrink T, Mogensen HS, Stene MC, Morling N

13. David J.Walsh,1 M.S;Amy C.Corey,1 B.S.; Robin W.Cotton, Ph.D.;Lisa Forman, ~ Ph. D. ; George L. Herrin, Jr. 2 Ph. D. ; Charlotte J.Word,1 Ph.D. ; and Daniel D. Garner, 1 Ph .D . Isolation of Deoxyribonucleic Acid (DNA) from Saliva and Forensic Science Samples Containing Saliva. 14. REFERENCE : Walsh, D. J. , Cor ey, A.C .,Cotton , R.W. , Forman , L . ,Herrin, G. L. ,Jr . ,Word , C J .and Garner , D. D . , "Isolation of Deoxyribonucleic Acid (DNA) from Saliva and Forensic Science Samples Containing Saliva, "Journal of Forensic Sciences, JFSCA, Vol . 37,No. 2, March 1992, pp.387-395. 15. Extraction of DNA from cigarette butts using proteinase forensicGEM 16. Charis Shepherd, Sallyann Harbison2 and David J. Saul 11 ZyGEM Corporation Ltd, Waikato Innovation Park, Ruakura Road, Hamilton, New Zealand. 2 Forensic division, Institute of Environmental Science and Research Ltd, Mt Albert Science Centre, Private Bag 92021, Auckland, New Zealand. 17. Laboratory experiments in Microbiology by M .Gopal Reddy, D.V.R. Saigopal, K.V. Mallaiah. 18. Crime Scene Management by Dr. M.S. Rao, Dr. B.P. Maithil, K.V. Ravi kumar.

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8. Forensic DNA Extraction Kit for Extraction of Genomic DNA from Biological Samples* 9. A Simple Automated Instrument for DNA 10. Extraction in Forensic Casework 154
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