Assessment of amplicons in the DNA from boiled tissue by PCR and AP-PCR amplification

Genetic Analysis: Biomolecular Engineering 12 (1995) 57-62

ELSEVIER

Biomolecular Engineering

Assessment of amplicons in the DNA from boiled tissue by PCR and AP-PCR amplification D e e p i k a M o h a n b, K.B.C. A p p a R a o a, A p a r n a Dixit h, Sher Ali a* aNational Institute of Immunology, .4runa Asaf .41i Marg, New Delhi, India bDepartment of Zoology, University of Delhi, Delhi, India Received 8 March 1994; revision received 21 April 1994; accepted 2 May 1994

Abstra~ Polymerase chain reaction (PCR) is a technique sensitive enough to amplify small DNA fragments a billion-fold. The generation of amplicons either by PCR with a set of oligo primers or by arbitrarily primed AP-PCR with a single oligonucleotide primer is based on the availability of intact template and priming sites. With these approaches, it is possible to generate specific and random amplicons to assess the extent of damage to DNA caused by any of the physical, chemical, or environmental factors. We report the amplification of sex chromosome and autosome specific loci in the buffalo (Bubalus bubalis) genome by symmetrical and APPCR performed on DNA samples isolated from the muscle tissues that were boiled (treated) for different lengths of time. No difference was noticed in the amplification profile of DNA cooked for various lengths of time. However, after Hinfl treatment, AP-PCR amplification of these DNAs revealed more bands on agarose gel than unrestricted samples. The successful amplification of the DNA samples isolated from the boiled tissues is attributed to the intactness of the amplicons. This suggests that despite storage for more than a year and subsequent heat treatment to the muscle tissues, the DNA remains a good substrate for PCR and AP-PCR amplification. Relevance of this work in the context of DNA probe technology is discussed.

Keywords: Ampficons; PCR; AP-PCR; Random primer

Introduction

The advent of polymerase chain reaction (PCR) technology has facilitated the amplification of DNA from a number of sources for its direct analysis [1]. The power of PCR is indisputable, a~ndamplification can be achieved even under the most stringent conditions. However, of all the components required for successful amplification intactness of primer annealing sites in the target substrate is the most important [2]. Several factors are known to degrade DNA samples leading to alteration or loss of the priming site,,;. Further, the presence of endonucleases within the tissues, their long-term storage, and excessive heat treatment are expected to damage the DNA, which may render the primer-template interactions ineffective for the generation of amplicons. How* Corresponding author.

ever, amplicons generated successfully from such samples would demonstrate the intactness of the priming sites in sheared DNA, and this may serve as a reliable parameter for evaluating DNA quality and origin. In DNA probe technology, a number of reports are available on the use and applications of PCR and APPCR [3-5]; but no report is available from boiled tissues or from cooked meat. The generation of specific amplicons by PCR for evaluating the DNA quality and origin would not indicate the intactness of the numerous random amplicons present in a genome. Thus, both PCR and AP-PCR need to be used to assess the specific and overall random amplicons. To ascertain specific amplicons, we have used sex chromosome and autosome specific primers for PCR amplification; and to evaluate the random amplicons, an arbitrary oligonucleotide primer was used for AP-PCR amplification. We believe that this approach may be applicable in deter-

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58

D. Mohan/ Genetic Analysis: Biomolecular Engineering 12 (1995) 57-62

Table 1 Details of the synthetic oligo primers used for PCR and AP-PCR Amplifications Serialno.

Prime~ [Req

~quen~s

1

BRY.I[7]

2

BOV97M [81

3

Bovine satellite [18] BOV. primers OATI5.2 [61

5'-GGATCCGAGACACAGAACAGG-3' 5'-GCTAATCCATCCATCCTATAG-3' 5'-GATCTrGTGATAAAAACrCrCTATGC-3' 5'-GATCACTATACATACACCACTCTA-3' 5'-TGGAAGCAAAGAACCCCGCT-3' 5'-TCGTGAGAAACCCrCACACTG-3' 5'-ACAGACAGACAGACA-3'

4

Si~ oftheamplified product (bp) 301 150 216 AP-PCR a

aThe AP-PCRrevealsseveralbands within the smear in the range of 0.2-1.9 kb (for further details, see Table 2).

mining the sex and origin of blood samples or tissues that have been cooked and in settling disputes related to forensic sciences, meat industry, and wildlife conservation programs. 2. Materials and methods

2.1. DNA isolation Buffalo (Bubalus bubalis) solid muscle tissue, stored for more than a year, was used for isolating the genomic DNA. Samples of I g muscle tissue from both sexes were boiled separately for 30, 45, 60, 75, and 90 rain in distilled water. All the boiled tissues along with the unboiled ones were processed for DNA isolation following standard procedure [6]. The DNA isolated from the boiled tissues was found to be - 3 kb or smaller (data not shown). 2.2. Amplification of Y-chromosome and autosome specific loci by PCR The Y-chromosome-specific primers BRY.1 and BOV97M [7,8] used for detecting specific loci are based on repetitive DNA sequences of cattle found to be conserved in all species of the family Bovidae (cattle, buffalo, sheep, and goat) [9]. Polymerase chain reaction amplification was carried out with normal and treated DNA (treated DNA refers to the samples isolated from the boiled tissues) samples using a set of Y-chromosome-specific primers, BRY.1 and BOV97M and an autosomal specific primer BOV (Table 1). Reactions were performed in 25-#1 volume containing 30-ng template DNA, 10 pM of each primer, 1.0 U Taq polymerase (Promega, USA), 2.5 mM MgCI2, 200 #M dNTPs, 50 mM KCI and 20 mM Tris-HCl (pH 8.3). The reactions were overlaid with mineral oil, heatdenatured at 96°C for 3 min, and amplified for 40 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 45 s, and extension of the primer at 72°C for 1 min using an automated thermal cycler (Perkin Elmer Cetus). On completion of the cycles, the amplified products were further incubated at 72°C for 5 min. The PCR products

were separated on 1.5% agarose gels (Pharmacia) at 20 V/cm for 16 h in TBE buffer. The gels were stained with ethidium bromide and photographed.

2.3. Amplification of random loci by AP-PCR A 15-base-long arbitrary primer (OAT15.2) based on a tetranucleotide repeat motif was used for AP-PCR amplification (Table 1). The origin of the probe sequences is reported elsewhere [6,10]. One set of AP-PCR amplifications were conducted on normal and treated undigested DNA samples, whereas another set of amplifications were performed on normal and treated DNA samples that had been digested with HinfI enzyme before their use as template for AP-PCR amplification. The reactions were carried out in 25 #1 volume containing 30 ng of genomic DNA, 2.5 mM MgCI2, 200 #M dNTPs, 10 pM primer, 50 mM KCI, 20 mM Tris-HCl (pH 8.3) and 1 U of Taq DNA polymerase (Promega). The reactions were subjected to low-stringency PCR with an initial denaturation at 96°C for 2 min 30 s followed by three cycles of denaturation at 94°C for 30 s, annealing at 36°C for 1 rain, and primer extension at 72°C for 2 min. The same reactions were further subjected to 35 cycles of denaturation at 94°C for 15 s, annealing at 50°C for 1 rain, and primer extension at 72°C for 1 rain 30 s. Finally, the samples were incubated for another 10 min at 72°C. After completion of the cycles, 6 #1 of the amplified products were electrophoresed on 1.5% agarose gel (Pharmacia) in TBE buffer at 20 V/cm, and bands were detected by staining the gel with ethidium bromide. 2.4. Southern blot hybridization Southern blot hybridization was performed to ascertain the total number of consistently reproducible bands detected by AP-PCR amplification. Products of APPCR resolved on 1.5% agarose gel were denatured in 0.5 N NaOH-0.15 M NaCl solution, neutralized with 3 M Na acetate (pH 5.5) for 20 min each and subsequently transferred onto Nylon membrane (Pharmacia LKB) with 20 x SSC following standard procedures [11]. The

D. Mohan I Genetic Analysis: Biomolecular Engineering 12 (1995) 57-62

59

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hn -,587 540 504 458 434 267 234 213 192 124

I r--.~

Fig. 1. Polymerase chain reaclion (PCR) amplification of sex chromosome and autosome-speeific loci in normal and treated DNA samples resolved on 1.5% agarose gel. All the lanes in A represent male DNA and in B female DNA samples. In both panels, lanes I and 2 represent normal DNA (N) and the remaining ones re:present DNA isolated from the tissue boiled for 30-90 min. In A, lanes !, 3, 5, 7, 9, and I I show the PCR-amplified product with Y-chromosome..specific primers BRY.I (301 bp) not seen in the corresponding lanes in B whereas, with autosome specific primers BOV, a 216-bp band is seen in both panels. Similarly, in lanes 2, 4, 6, 8, 10, and 12 of A, a 150-bp male-specific band is seen with another sex-specific primer BOV97M that is absent in the corresponding lanes in B. M is the molecular size marker, pBR322 DNA digested with HaellI enzyme.

oligonucleotide probe OAT15.2 was labeled following the method described earlier [6]. Hybridization was performed at 40°C for 16 h in 5x Denhardt solution, 5x SSPE, 10 t~g/ml Escherichia coil DNA as carrier DNA (sonicated and denatured), and 1% S.D.S. Filters were subsequently washed four times in 6× SSC at room temperature followed by two washes in 6× SSC for 15 rain each at 38°C. The filters were then exposed to X-ray film (Indu/India) with two intensifying screens (Cronex Lightning Plus, Dupont) at -70°C.

3. Results

Symmetrical PCR and AP-PCR amplifications performed on normal and treated DNA samples revealed sex-chromosome-specific and a number of autosomespecific isomorphic amplicons. With Y-chromosomespecific primers, normal and treated DNA samples both revealed discernible bands of 301 and 150 base pairs (bp) corresponding to BRY. 1 and BOV97M primers in the male sex whereas a 216-bp band corresponding to

0÷ 0+ 0" o" O~r~ 0 ~r,. 0o~0 t~ n ~O © tnI'~ 0~ N, I I I l l I I I I [ I I I I I I 1 2 3 4 5 6 7 8 9 10111213141516 I

I'

[

I

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M 2-Okb

E.O-5kb

Fig. 2. AP-PCR-amplified loci of HinfI digested buffalo genomic DNA with a single arbitrary primer OATI5.2 resolved on 1.5% agarose gel. Lanes 1-10 represent DNA samples isolated from tissues boiled for 30 to 90 rain. Lanes 11-16 contain normal DNA samples. Note the discernible isomorphic loci in the range of 0.2-1.9 kb in all the lanes (treated and normal). Compared with normal ones, treated DNA samples show a few more resolvable bands. This difference was not noticed when undigested DNA samples were used for AP-PCR amplification (not shown). M is the fragment size in base pairs.

60

D. Mohan I Genetic Analysis: Biomolecular Engineering 12 (1995) 57-62

bovine satellite specific primers BOV was seen in both sexes (Fig. 1). In symmetrical PCR, no difference in the amplification products was noticed between normal and treated DNA samples with any of the primers. In the AP-PCR amplified products, a difference in the overall band pattern between the digested normal and digested treated DNA samples was observed. In amplified digested-normal DNA samples, a maximum of 12 bands was observed whereas in the amplified digested-treated DNA samples, a maximum of 16 bands in the range of 0.2-1.9 kb were observed (Fig. 2). PCR amplification with a single primer performed under reduced stringency conditions can give rise to a finite number of bands that can be resolved on agarose gels. Nevertheless, these discernible bands may not be the true representative of the randomly amplified loci (amplicons) as false priming may contribute to PCR products [12]. Thus, to detect consistently reproducible amplicons, Southern blot hybridization of AP-PCRamplified products was performed using an arbitrary primer OAT15.2 as a probe (Fig. 3). Southern blot analysis did not reveal all the amplicons that were detected on the agarose gel before hybridization (not shown). The details of all the AP-PCR amplified loci detected after hybridization in normal (control) and treated DNA samples with and without Hinfl digestion are given in Table 2. It was noticed that with a single arbitrary primer OAT15.2, AP-PCR amplification of Hinfl digested DNA samples for three cycles under reduced stringent conditions followed by 35 cycles under high stringent conditions gave rise to a number of discernible bands characteristic of a particular species. In this context, we included goat DNA along with normal buffalo DNA samples for the AP-PCR amplification and detected a genome-specific band pattern both before (not shown) and after Southern blot hybridization (Fig. 4). This observation was further substantiated by an independent set of AP-PCR reactions where four different species-cattle, sheep, goat, and human, revealed genome-specific amplification patterns (not shown).

d o

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4

5

6

7

8

9

O~

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10 11 12 M

~2.3 -2"0

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Fig. 3. Autoradiogram of AP-PCR amplified loci after hybridization with primer OATIS.2 as probe. Undigested DNA samples were used for amplification. Lanes I and 7 contain normal DNA (N) and the remaining ones represent DNA samples isolated from tissues boiled for 30-90 min. A maximum of four isomorphic bands in the range of 450-830 bp were recorded. In lanes 3, 5, 6, and 8, the 830-bp band is not discernible owing to less amount of DNA loaded. M is the molecular size marker, lambda DNA digested with H/ndIIl enzyme.

Table 2 AP-PCR amplified Loci in Buffalo Bubalus bubalis and goat a Caproa hircus genomes detected by OATi5.2 primer used as probe in Southern blot hybridization Source of target DNA

Undigested/digested

No. of loci detected

Approximate size range of the bands (bp)

Normal buffalo tissue sample

Undigested Hinff digested Undigested Hinfl digested HinfI digested

4 7 4 3 8

450-830 350-930 450-830 470-670 350-1100

Treated buffalo tissue sample Normal goat tissue sample

aGoat DNA was used with buffalo DNA samples to compare the overall band pattern between the two species (see the text for methodological details).

D. Mohan I Genetic Analysis: Biomolecular Engineering 12 (1995) 57-62

OLnO ,~OOLnC)~O I i

© i

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I

n I

,,-

I

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--N-I I I

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Fig. 4. Autoradiogram of AP-PCR amplified loci detected after Southern blot hybridization. Hin!['l-digested genomic DNA samples were used for amplification (lanes: buffalo 1-14: and goat [G] 15-16) and hybridized with the primer OAT 15.2 as probe. Lanes 1-10 represent DNA samples isolated from tissues boiled for 30-90 min whereas lanes 11-16 represent normal DNA. Note some common isomorphic bands in treated and normal buffalo DNA samples. The goat samples reveal some uncommon isomorphic bands not seen in the buffalo samples. M is the molecular size marker, lambda DNA digested with HindIII enzyme.

4. Discussion

The PCR and AP-PCR techniques amplify and reveal different regions of the genome and thus complement each other. In the past, several attempts have been made to examine the suitability of DNA samples recovered from different evidential materials by using conventional restriction fragment-length polymorphism and by PCR in some cases [13]. However, there seems to be no information available on PCR amplification performed on the DNA samples isolated from boiled tissue or cooked meat. Similarly, arbitrary primers have not been used to establish the DNA source nor has the sex of any animal species been determined from boiled or cooked tissue. Therefore, the use of an arbitrary primer for AP-

61

PCR amplification together with the chromosomespecific primers for symmetrical PCR seems to be a better approach to demonstrate the availability of the overall amplicons in the genome. In the present study, successful PCR amplification of treated DNA samples with sex chromosome and autosome-specific primers indicates that specific priming sites remain intact even though the tissue was stored for more than a year and was subsequently boiled for a time range from 30 to 90 rain. This is a reasonable time for conventional cooking, and if this is taken as a reliable parameter, it then follows that the sex of an animal may be determined by the PCR approach even by using cooked meat or boiled tissue samples. Similarly, we compared the AP-PCR-generated amplicons from normal DNA with the samples isolated from the boiled tissues. It was expected that the exposure of tissue samples to excessive heat before DNA isolation may change the available priming sites especially when the DNA has been stored for more than a year and such samples compared with normal ones may show a difference in the AP-PCR amplification profile. How ever, no difference was noticed in the AP-PCR amplified products of normal and treated DNA sampies. Further we wanted to assess the amplicons in the Hinfl digested normal and treated DNA samples. It was noticed that AP-PCR amplification of digested normal and treated DNA revealed more bands compared with undigested ones. The DNA isolated from the boiled tissue samples was sheared, and the fragment size was found to be < 3 kb. Successful amplification of such DNA suggests that despite heat treatment of tissues, enough amplicons remain intact for AP-PCR. AP-PCR amplification has been used for genomic DNA fingerprinting and for species identification in a number of instances [14,15]. In the present study, following the AP-PCR approach, we used twelve random DNA samples from buffalo to test if the primer can generate DNA fingerprints. Surprisingly, several species-specific monomorphic bands were detectable instead of a DNA fingerprinting pattern. Preliminary studies in our laboratory using normal goat, sheep, cow, buffalo and human DNA indicate that AP-PCR using the primer OAT15.2 reveals distinct species-specific amplicons on amplification (not shown). For AP-PCR oligo primers can be chosen arbitrarily without prior knowledge of the genomic sequence of the species to be investigated [4,15]. We observed that digested DNA samples used for AP-PCR amplification compared with undigested ones reveal a greater number of bands. Similar observations have been made by CaetanoAnolles et al. [5]. However, it has been reported in another study that digestion of DNA before amplification reduces the total number of bands but increases the intensity of some bands in comparison with undigested samples [16]. Though the reports are conflicting, we

62

D. Mohan/ Genetic Analysis: Biomolecular Engineering 12 (1995) 57-62

believe that digested DNA, due to smaller size, probably facilitates the primer annealing on additional sites and hence gives rise to a greater number of bands. The discrepancy detected in the number of bands in the APPCR amplified products before and after DNA hybridization is possibly due to the specificity of the oligonucleotide probing. Oligo probes, under stringent conditions are known to hybridize with target DNA only when perfectly matched sequences are present [17]. Thus, the amplicons not detected after Southern blot hybridization were the ones that did not have sequences exactly complementary to the oligo probe. Though, we have not used DNA samples isolated from boiled tissues of human or other species, it is expected that the DNA isolated from the remains of burnt bodies or from the samples exposed to excessive heat may be used for determining the DNA origin and the sex of the individual. For settling disputes, this approach may be applicable in meat industry for determining meat origin and adulteration (meat from two different species in bulk supply) and for sexing tissues from cold storage or cooked meat. Further, this approach may be useful in wildlife conservation programs for establishing the identity of animals and their sex, particularly for species that are fast becoming extinct due to the ever increasing menace of poaching. Another advantage of this technique is that a small amount of genomic DNA (30 ng), which may be sheared or partly degraded, can be used for analysis. In conclusion, the data suggests that PCR and AP-PCR approaches would be useful for determining the origin of the animal from the boiled tissue samples.

Acknowledgements The work was supported by a core grant to the National Institute of Immunology from the Department of Biotechnology, New Delhi, Government of India. D.M. is a recipient of a Junior Research Fellowship from the Council of Scientific and Industrial Research, New Delhi.

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