DNA FINGERPRINTING: IT’S DEBUT IN INDIA. ISOLATION OF HIGH-MOLECULAR-WEIGHT DNA.

Isolation· of High-molecular-weight DNA

S. KARUTIIA PANDIAN,

M. CHANDRA

SEKAR, KS. ANNAPOORANI,

B.

NAZIRUDDIN,

P.

CIIANDRA SEKHARAN, C. DAMODARAN

Forensic Scienccs Department, Madras -600 004, India

DNA - PARMAKİzİ HİNDİSTAN'DA İLK ADıM

Yüksek Molekül Ağırlıklı DNA'nın İzOlasyonu

Özet

DNA parmakizi uygulamalarının ilk ve en önemli basamağı incelenen biyolojik örnekteki DN A molekülünün sağlam bir şekilde izolasyonuduL Izole edilen yüksek molekül ağırlıklı DNA'nın daha ileriki aşamalarda gerçekleştirilecek restriksiyon ve blottransferine yetecek miktarda ve uygun saDıkta elde edilmesi başlıca hedeftir. Hindistan'da DNA parrnakizi yönteminin adli uygulamalara ışık tutmak üzere kullanılması amacıyla başlatılan çabalar 1986 yılına dayanır. Uğraşların ilki tartışmalı bazı babalık belirtimlerinde, insan katı örneklerinden yüksek molekül ağırlıklı DNA'nın izolasyonu olmuştır. Bulguları sunulan bu çalışmada Ci/I ve ark.(1985)'nın değiştirilmiş yöntemine göre kan örneklerinden izole edilen DNA molekülünün elde edilme verimi ortalama 27 mg/ml olup, saflığı 1.8 ( OD260 / OD 280 ) bulunmuş, molckülün sağlamlığı agaroz jel elektroforezi ile araştırılmış ve

bulguların literatürdeki verilere uyduğu saptanmıştır.

Summary

The first and the critical step in DNA fingerprinting exercise is to isoIate from the biospecı 'ıcn concemed, the genomic DNA intacı. High-molecular-weight DNA thus obtained should be quantitat;vc and pure enoıigh for processing thenceforıh through restriction and blot transfer. By the way inıroducing the practice of DNA fingerprinting to Indian science espccially fdr forensic utility, work was initiated in ı 986; this communicaıion based on such attenipt presents data on the recovery of high-molecular-weight DNA from human blood samples including some from cases of dispu1ed parentage.

Kcy words : DNA-jingerprinting - lIigh molecular weight DNA isolation - Paternity

14

s.

KARUTIIA PANDIA~ ct al

INTRODUCTION

The concept of idcnLifying in humans individualistic

features at the molccular lcvcJ

through geneLic markers, set to roll at the dawn of this century, has

been,

over the past

decades, weıı elaborated in every

aspect-content, scope and appjication. Contributions to

this beneficial "metamol1;hosis" have come from ncver-ceasing cliscoveries of more and

more

genctic polyınorplıism espeeially of proteins/enzymes. To

this eclifice,

a

totally

new dimension

from a deeper perspeetive was given in 1985 by leffreys (1) w

h

o

reporl-c

d hypervariable

"ıninisatellite" areas comprising inheritable variations in the type and

number of arrays

of

repetitive base sequences in human genomic

DNA. This "DNA

fin-gerpriming"

has not only

apparently

overshado\VeCı all the known genctic

systems but

also

revolutionizeCı

many disciplines of basic and applicd sciences, as is evidcnt by the

ever-growing number of publications related to fundamental genetics, population genet

-ics,

medical genetics, animal genetics,

demography, epiclemiology, taxonomy,

oncolo-gy, psychiatry, diagnostic medicine ancl forensic practiee

i

nclucling parentage

detem1İna­

tion

(2-37). Arather in[ormal apex

bocly

on forensie

hemogenetics

at the global

level

namely the

International Society

for

Farensic Hemogenetics, has also recommended

through

wiLh

pragmatic caulion,

the use of DNA fingerprinling

(}ö). Wİlh the proven

possibility of obtaining

in

great quantities mu1tipl copies of trace amounts (as

usuaııy

is the case

i

n many

forensic situations) or DNA through polymerase chain reaction

(39-41), the

status of DNA fingerprinting forensic arena is cenainly at its zenith.

I

n

further-ance of this, there are available nonradioactive/safer probes (key "reagents") (42-46) for

easy introduction of DNA fingerprinting İn operational forensic

laboratories

where

oth-crwise the infrastructure for handling radiolabeled probe s does not usually

exisı<;.

Any atteınpt to study D

N

A

polymorphisın

u

s

ing "minisatellite" probes requires

first

of

all isolation from

biospecimen

of high quality high-molecular-weight DNA in suffi

-cient quantity. This is sub

j

ccted

to many variables like the isolation

method, nature of

tissue,

storage effecı

an

d in

t

he case of bloodstain the behaviour of the substratum of

de-posit (46-55).

It is in this context that data is presented in this article on the eharacter

i

-zation of fingerprinting-worthy intilct DNA from

human blood saınplcs, which fonned a

part of the

authors' pioncering

i

nitiative in the Indian contextthat too from forensic

per-spective.

MATERIALS AND METHODS

Samples

Iluman venous blood samples were coJlcetcd in ACD solution from a ıotalaf 40 individuals (l ı, be10nging to three differcnt eases of parentage disputc; 14, from a large pedigree; 15, random donors).

For isolating high-molccular-wcight DNA, the blood samples wcre proccs~ed using the method described by Cill ct al (4) with suitable modifications.

Yie/d and Quali/y of DNA

Optical density measurements at 260 nm and 280 nm were taken for working out the yield (OD2Go ) and purity (ODZGO /OD2so).

Res/rie/ion and Blo/-/ransfer

IIigh-molceular-weight DNA (inıactness cheeked by agarose gel cleetrophoresıs) isolated from blood was subjected LO restrietion cleavage by the endonuclease Eco RI/Hind III with appropriaıe incubation medium; agarose gel elceırophorcsis and Somhem blot transfer were subsequently earried

out. The proeedure of Gill et al (4) was adopted with modifications.

RESULTS AND DlSCUSSION

Table i

sets out the figures accounting for the yielel of

high-molccular-weight DNA

[rom human bloocl (40

samplcs)

and the purity of

such

isolates.

Tablc i. Data on the qualiıy of DNA isolated from 40 samplcs of human blood

DNA yicld DNA p\lrit~

(ıng/ml) (ODZGO j OD280 )

Min Average Max Min Average Max

22 27 30 1.56 1.8 1.9

The

isolated

DNA was suhjectcd to suhmarine gel electrophoresis along

with

le

phage DNA for comparison; the pattem as vicwed unde

r

VV arter ethidium

bromide

staining

is

illustrzıteel

in Figure 1.

Figure 2 represents the electrophoretic demonstration on the gel, of the

r

estriction-clcaved DNA isolates. The

same

gel when stained arter Southcm blot showed

no

evİ­

elcnce

of

DNA thereby confinning total transfer to membrane.

On the

whoıC

il is evielent that in

the

authors' hancls, the exerc

i

se of

obtaining

from

human blood

goocl

quality DNA for subsequcnt fingerprinting has been

f

ruitful.

The

yicld as well as the purity (Table I) are in gooe! agreement

with

if not

better

than those

reported by others (19,48,54). The clectrophoretic demonstrations in

subsequent

stagcs

(Figures 1-2) also bem encouraging proof. Data on the DNA typing using differen

t

16 S. KARUT[]A PANDIAN ct al

FigllfC ı. Agarosc (0.7 %) gel clectrophoresis dcmonstrat.ing the intactness of high m olecular vieight DNA isolated from human blood samples shown along with I, DNA. Lanes 1-4: human DNA; lane 5: Hind III-digesıcd le phage DNA.

Figure 2. Agarose (1 %) gel eJcctrophoresis

showing Hind III restricted [ragments of le D:-\A (lane 1) and human DNA (tane 2).

A ekli owl cd gem c il ts

The authors thank:- Prof.Dr. K. Dharmalingam and Dr. T. Rajapandi of :\1K University, Madurai; Prof.Dr. K. Jayaraman, Anna University, Madras; Prof.Dr. A.J. Jeffrcys, Leicester University, UK; and, Dr. D.J. Wcrrett, Central Rescarch and Support Establishment, Home Office Forensic Science Service, UK - for their assistancc/collaboration.

REFERENCES

Jcffreys, A.J., Wilson, V., Thein, S.L. (1985) IIypervariable "minisatellile" regions in human

DNA. Na/ure, 314,67-73.

2 Jeffreys, A.J., Wilson, V., Thein, S.L. (1985) Individualspecific "fingerprints" of human DNA. Na/ure, 316, 76-79

3 Jeffrcys, A.J., 13rookfield, J.F.Y., Semeonoff,R. (1985) Positive iden1ifieation of an immigra-tion test-ease nsing hnman DNA fingerprinıs. Na/ure, 317, 818-819.

4 Gill, P., Jcf[reys, A.J.,· Werrett, D.J. (1985) Forensie application of DNA "fingerprints". Na-turc, 318, 577-579.

5 13ohm, L., Taitz, J. (1986) The DNA [ingcrprint: a revolutionary forcnsic identification test. S.

Afr. Law I., 103, 662-669.

6 Thein, S.L., Jc[[rcys, A.J., macklock, ILA. (1986) Identification of post-transplant cell popn-lation by DNA fingerprint analysis. Lanee/, ii, 37.

7 Jeffreys, A.J., Wilson, V., Thein, S.L., Weatherall, D.J., Ponder, 13.A.J. (1986) DNA

"fıııgcrprplls" and segregation analysis of mnltiple markers in human pcdigrees. Am. 1. l1um. Gene/., 39, 11-24.

8 Sensabangh, G.F. (1986) Forensic biology-Is Tecombinant DNA technology in its future? I. Forensie Sci, 31, 393-396.

Madufai, Paper 102.

II Werrett, D.J. (1987) DNA fingerprirıting. Int. Criminal Pohce Rev., 408, 21-25.

12 Gill, P., Lygo, J.E., Fowler, S.J., Werrett, D.J. (1987) An evaluation of D:'-fA fingerprinting for forensic purposes. Elecıroplıoresis, 8, 38-44.

13 lIonma, M., ıshiyama, 1. (1987) DNA fingerprinıs: its importance in forensic medicine (i) -application to paternity testing by minisatellite DNA probes. Jpn. J. Legal Med., 41, 236-24 ı.

14 lloltz, J., ülek, K., Higuchi, M. (1987) Forensic use of DNA fingerprints. Beitr. Gerichil. kfed, 45, 5-10.

15 Scherer, S., Steven s, D.A. (1987) Application of DNA typing mcthods to epidemiology and taxonomy of Candida species. 1. Clin. Microbiol., 25, 675-679.

16 Jcffrcys, A.J. (1987) lIighly variable minisatellites and DNA fingerprints. Biochem. Soc. Trans., 15, 309-317.

17 George, M., Cochaux, P., Lequarre, A.S., Young, M.W., Vassart, G. (1987) DNA fingerprinting in man using a mouse probe rclated LO part of the Drosophila per gene. Nucleic Acid Res., 15, 7193.

IS Gill, P., Werrett, D.I. (1987) Exclusion of aman chargcd with murder by DNA fingerprinting. Forensic Sci. Inı., 35, 145-148.

19 Jcffreys, A.J., Morton, D.B. (1987) DNA fingerprinıs of dogs and cats. Animal Genel.,18, 1 -15.

20 Ilill, W.G. (1987) DNA fingerpriııts applied to animal and bird populations. Nalure, 327, 98-99.

21 Wetton, J.li., Carter, R.E., Parkin, D.T., Walıers, D. (1987) Dcmographic study of a wild house sparrow populaıion by DNA fingcrprinting. Nalure, 327, 147-149.

22 Burkc, '1'., Bruford, N.W. (1987) DNA fingerprinting in birds. Naıure, 327, 149-152. 23 Bohm, L. (1987) Saıellite D:\:A comes to the fore. S. AJr. J. Sci., 83, 243-244.

24 Van IkIden, P.D., Wiid,

u.r.

(1987) Application of DNA fingerprintİng lo the investigaıion of cell line genotypc. S. AJr. J. Sci, 83, 244.

25 üdelberg, S.J., Demers, D.B., Weston, E.II., Hossaini, A.J. (1988) Establishing patemity using minisatelliıe DNA probes when the putalive father is unavailable for ıesting. 1. Forensic Sci., 33, 921-928.

26 hhiyama, L, Yoshini, T., IIonma, M., Mukaida, M., Yamakuchi, T. (1988) DNA fingerprints: The significance of examining DNA polymorphisms of placental ıissues for the purpose of paıemity detenIıinatİon during the early stages within the first trimester. Z. Rechlsmed., 99, 241-248.

27 Tokahashi, T. (1988) Human genetİc diagnosis by DNA probes. Bio fnd., 5, 215-220. 28 Tokahashi, T. (1988) Human genelic diagnosis wiıh D\'A probes. Bio Ind., 5, 284-289. 29 Sakai, T., Yoncda, H. (1988) Geneıic engineering and psychiatry. Sei.~hin Igaku, 30, 291-297. 30 IIumpries, S., Taylor, R., Jeenah, M., Seed, M. (1988) The use of rDNA techniques for ıhe

diagnosis of familial hypercholesterolaemia. J. Inheriıed Melab. Dis. (E), 11, 29.

31 Karutha Pandia~, S., Chaııdra Sekar, -'.1., Anııapoorani, K.S., Nazirııddin, B., Chandra Sekheran, P., Damodaran, C. (1989) DNA polymorphism/fingerprinting - the firsl forensic attempt in India. Adli Tıp Derg., 5,67 -72 .

32 Devor, LJ., Ivanovich, A.K., IJickok, J.M., Todd, R.D. (1988) A rapid method for confirming ecU line identit)': DNA "fingerprinling" with a minisatellite probe from Ml3 bactcriophage. B ioıechniques, 6, 200-202.

33 1'larx, J.L. (I98S) DNA fingerprinting lakes the witncss stand. Science, 240, 1616-1618. 34 Landegrcn, U., Kaiser, R., Caskey, C.T., Hood, L. (1988) DNA diagnostics - molecular

S. KARUTlIA and amomation. Sciu2(c.

(1988) Genctic fingcr- rn New Scienıisı, 117 .

. (1988) DNA fingerptlnr ;iogisı, 12, 2-8.

31 Wdl>, R.". (1988) DNA fingerplliıııng. ln Genume Analysis, A Praclicai APplOcich (K.E. Davics, cd) pp.l53 -170, IRI. l'n:ss, Oxford.

38 Anonymus (1988) DNA recommcndation of the DKA commitıcc, International Socicty for Forcnsic ıraemogcııetics (ISE\} ISFII Newsleııer, Dcccmber '88.

39 Marx, n~. (1988) MlIJıiplying gcncs by leaps and bOlinds. Science, 240, 1408-1410. 40 Saiki, R.K., G yllcııs ten, e.B., Erlich, ILA. (1988) Thc polymerase chain reacıion. In Genome

Analysis, A Praclical Approoch (K.E. Davics, cd) 1'1'.141-152, lRL Press, Oxford.

41 lliguchi, R., von BcroldiııgCIl, CIL, Scnsabaugh, G.F., Erlich, ıLA. (1988) DNA typiııg from

siıır.k h"j". Nalurt, 332, 5,13-54(' .

J.A., Batki, A., Ilyndj, (1985) Enhanced chemilumı, ('2C,cıı1

dc:!Getion of DNA dot-h) AnaL. Biochem., 151,

I .. L, Kcllogg, D.E., ıson, Cıl., Erlich, I!

M 13 probe> for gencıic eaLİon ıo ilLA class ıı

83, 9085-9089.

44 '-/., Morisaki, T, Fuji;. iJS8) A simI'le huma;'

biotinlabcllcd probe;. Med., 42, 351-353.

D.D. (1988) Thc use oj probes in parentage

labcling and non-Iu'2ic cxlraetion. Eleclrophoresis, 9, 359-368.

46 Chan, V.T.W., Fleıııing, K.A., McGee, J.O'D (1988) SiıııulLancoııs extraction from clinical biopsics of high-ıııoJccular-weight DNA and RNA: comparaliye characıcrization of biotinylatcd and 32p -labellcd ımıbes on Southern and Northem blots. AnaL. Bioehem., 168, 16-24. 47 Casilio-Lconardo, E, :lachary, A.A., Klinger, K.W. (1987) Extraction of DNA from solid

ıissuc. 1. Clin. Lab. Ana/. I, 146-150.

48 Gustafson, S., Proper, J.A., Bowie, E.J.W., Sommer, S.S. (1987) Parameıers affeeting the yicld of DNA from huıııan bloo,!. Anal. Biochem., 165, 294-299.

L., IIoar, D.I., ııoıroO'd, Ilodes, M.E. (1987)

;Lorage of blood and iso!;,lcd OIck, K. (1988) Use of

193-198.

",!ing, I., Joseplıi, E., in eadavcr ıissues. Bei!r

TeifCı-Grcding, .J.

ıntegri!y of DNA. Am.

markers in tracc anal:;,;: iS,

, E. (1988) Stability 46, 199-202.

Liebhardt, E. (1!L

high-molccuJar-wcight DNA from a corpsc which had be en under water for 14 days. Bei/r.

GerichıL. Med, 46, 203-206.

53 Bahnak, B.R., Wu, Q.Y., Coulombcl, L., Drouet, L., Kerbirioıı-l\abias, D., Meyer, D. (1988) A siınple and efficicııı method for isolating high mokeular weight ])0[A from mammalian speflll. Nuc/eic /1cids Res., 16, 1208.

54 Miller, S.A., Dykcs, D.D., l'oJcsky, II.F. (1988) A simple salting out rroeedure for extracting DNA from hııman Iluekated eclis. Nuc/eic Acids Res., 16, 1215.

55 Kalbc, J., Kuropka, R., :\1cycr-Stork, L.S., Saıııer, S.L, Loss, P., Ilenco, K, Riesncr, D., Bcrndt, ıl. (1988) Is!> :ıi ;,<11 C1"I;!i! Icrizaıion of high-molecular fruııı

Biol. Chem. Jfoppe-S"y!cr, ,,'1 1

Rcpriııts rcqui'st C Damodaran Forcnsic SCiCIlCl'., Madras - 600