Enzymatic synthesis of DNA probes complementary to a human variable number tandem repeat locus

ANALYTICAL

BIOCHEMISTRY

179,280-283

(1989)

Enzymatic Synthesis of DNA Probes Complementary Human Variable Number Tandem Repeat Locus

to a

Sher Ali and R. Bruce Wallace Department of Molecular Biochemistry, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, California 91010

Received

August

4,1988

Both cloned and synthetic DNA probes complementary to human variable number tandem repeat (VNTR) loci have been used to detect restriction fragment length polymorphism. In this report, we describe an approach for the enzymatic synthesis of a DNA probe complementary to one VNTR locus. The probe is produced by annealing short synthetic oligonucleotides comprising single repeat units and enzymatically ligating them into a polymeric DNA probe. In Hi&I digests of human genomic DNA separated by agarose gel electrophoresis, this ligated oligonucleotide probe (LOP) detects multiple polymorphic loci in the range of 3-23 kb producing highly informative DNA fingerprint patterns when different individuals are compared. The hybridization pattern is very stable even under highstringency wash conditions. The LOP is more easily generated than cloned VNTR probes and is totally synthetic, avoiding problems associated with cloned probes including bacterial growth and maintenance as well as in vitro labeling. 0 1989 Academic Press. Inc.

The human genome contains a significant amount of repetitive DNA. Repeat sequences are either dispersed throughout the genome (1) or arranged in the form of long arrays of short tandem repeat units (2). The tandemly repetitive sequences are often highly polymorphic due to variation in the number of copies of the repeat units (3,4). The regions of the genome containing polymorphic tandem repeated sequences have been termed variable number tandem repeat (VNTR)’ loci (5) or minisatellites (6). Probes complementary to the tandem 1 Abbreviations used: VNTR, variable number tandem repeat; LOP, ligated oligonucleotide probe; COP, conventional oligonucleotide probe; kb, kilobase pairs; SSC, 0.15 M NaCI, 0.015 M sodium citrate; SSPE, 0.18 M NaCI, 10 mM sodium phosphate, 1 mM EDTA, pH 8.0; SDS, sodium dodecyl sulfate.

280

repeat unit are useful for detecting length polymorphism in VNTR loci (5). In earlier studies, polymorphisms were detected using cloned fragments as hybridization probes (6-8). More recently, synthetic oligonucleotides have been used for studying polymorphisms. In these loci, we have demonstrated fingerprinting DNA by oligonucleotides specific for simple repeats (9). Furthermore, with the appropriate oligonucleotide probes, a locus-specific hybridization pattern has been obtained with several VNTR loci. This approach has been employed to monitor the successof engraftment following bone marrow transplantation (10). Studies on sequence polymorphisms in VNTR loci have found a wide range of applications including constructing genetic linkage maps (ll), resolving paternity disputes and proving relatedness in immigration cases (12), identifying the origin of DNA in forensic medicine (13,14), determining twin zygosity (15), and fingerprinting DNA in domestic animals (16). Thus, DNA hybridization probes based on repeat elements whether cloned or synthetic DNA are of great value. Therefore, any attempts toward improvement of these probes will be useful. In view of this, we have developed an approach by which a VNTR probe can be enzymatically synthesized in vitro. The protocol is based on the synthesis of two short oligonucleotides each partially complementary to the other in such a way that when annealed they form a long double-stranded DNA molecule with nicks which can be ligated using T4 DNA ligase. In this way, the ligated probe becomes a mixture of various lengths of monomer units. Prior to the annealing, one of the oligonucleotides may be radiolabeled at the 5’ end using T4 polynucleotide kinase. After the annealing and enzymatic ligation, the polymeric product can be used as a hybridization probe. MATERIAL

AND

METHODS

Origin and Design of the Oligonucleotide Sequences The sequences used in this present experiment are related to the 33.15 locus originally described by Jeffreys 0003-2697/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

ENZYMATIC

SYNTHESIS

et al. (6). This locus is a tandem repeat of a 16-nucleotide-long sequence the consensus of which is (5’GAGGTGGGCAGGTGGA). Two Is-base-long oligonucleotides each complementary to a permutation of the repeat unit (Fig. 1A) were synthesized using an automated DNA synthesizer (Applied Biosystems) and purified by polyacrylamide gel electrophoresis and HPLC using a PRP reverse-phase column (Hamilton).

OF

DNA

A 3315-16.1

5'

X315-16.2

S’OH TCCACCTGCCCACATC 1 c

1

ANNEAL

I-

DNA LIGASE

Approximately 10 pmol of each oligonucleotide was labeled at the 5’ terminal hydroxyl group using [T-~~P]ATP (20 pmol, NEN, 7000 Ci/mmol) and 5-7 units of T4 polynucleotide kinase (New Bio Labs) in a lo-p1 reaction volume containing 67 mM Tris-HCl, pH 8.0, 10 mM Mg and 10 mM dithiothreitol. The kinase reaction (OAc),, was carried out at 37°C for 45 min. For conventional oligonucleotide probe (COP) hybridization, the labeled oligonucleotides were then separated from the [T-~~P]ATP by column chromatography over DE 52 cellulose (Whatman) as described earlier (17).

Annealing

pCAGGTGGAGA~GT;GG +

of the Oligonucleotides

Radiolabeling

281

PROBES

5' . . ..pCAGGTGGAGAKGTRGGCAGGlGGA-GAKGTRGGCAGGTG&MAKGTRGG

B COP

LOP

of the Labeled Probe

One of the two oligonucleotides (Fig. 1A) was labeled as described above and, without subjecting it to chromatographic separation over DE 52 cellulose, the solution (10 ~1) was boiled for 5 min in order to inactivate the enzyme. A l-p1 sample was reserved for polyacrylamide gel electrophoresis, the remaining 9 ~1 was combined with 20 pmol of unlabeled, partially complementary sequence (Fig. lA), and the annealing reaction was carried out in 25 ~1 of 40 mM Tris-HCl, pH 7.5, 20 mM MgC12, and 50 mM NaCl at 65°C for 4-5 min. The annealing reaction was then cooled slowly to ambient temperature over 15-20 min.

Enzymatic

Ligation

of the Annealed

Probes

After annealing, the enzymatic ligation was carried out at 4°C for 15 min in a total volume of 30 ~1 in a buffer containing 50 mM Tris-HCl, pH 7.8, 10 mM MgC12, 20 mM dithiothreitol, 1 mM ATP, 50 pg/ml bovine serum albumin, and 15-20 units of T4 DNA ligase enzyme (New England Bio Labs). Addition of polyethylene glyco1 did not improve ligation efficiency or produce longer products.

Polyacrylamide

Gel Ekctrophoresis

The kinase and ligase reactions were electrophoresed on 6% polyacrylamide gel under denaturing conditions using 7 M urea, 89 mM Tris, 89 mM boric acid, 2 mM EDTA at 2000 V on a sequencing gel apparatus (21 X 50 cm). Approximately 20,000 cpm of probe in a total volume of 3 ~1 of 95% formamide, 20 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cynol FF were elec-

-n

A schematic illustration of the 3315-16.1 and 3316.16.2 sequences, their annealing, and their ligation into a polymerized product (LOP). (B) Autoradiogram of the Is-base-long conventional oligonucleotide probe (COP 3315-16.1) labeled at the Fi’hydroxyl

FIG. (COP)

1.

with 32P using T4 polynucleotide kinase and electrophoresed on 6% polyacrylamide gel under denaturing conditions. LOP is the ligated oligonucleotide probe. The polymerized products are multiples of the monomer (n = 16). Note that the substrates and products are mixtures of different sequences: K, G and T; R, A and G; M, A and C; and Y, C _and T.

282

AL1

AND

WALLACE

COP

trophoresed. After the electrophoresis, the gel was dried and exposed to X-ray film (Kodak X-AR) for lo-15 min at room temperature. After analysis by gel electrophoresis the LOP was used directly for hybridization without further purification.

DNA Isolation, Restriction Digestion, Electrophoresis, and Hybridization

1234

LOP 5

12345

Agarose Gel

DNA was isolated from the blood cells of unrelated individuals according to the methods described earlier (9). DNA (4-5 pg) was digested with HinfI restriction enzyme (New Bio Labs) according to the supplier’s specification and the digested DNA was electrophoresed in 0.7% agarose gel (Seakem ME) in the recirculated buffer (40 mM Tris, 12 mM sodium acetate, 2 mM EDTA, pH 8.3). For size estimation, X lambda DNA digested with Hind111 was included in the gel electrophoresis. After the electrophoresis, the gel was stained with ethidium bromide and photographed under ultraviolet light. The gel was then dried for 1 h at room temperature on a vacuum gel drier (Bio-Rad) and for another 1 h at 60°C. Prior to the hybridization, the DNA within the gel matrix was denatured by soaking the gel in 300 ml of 0.5 N NaOH, 0.15 M NaCl at room temperature for 20 min and then neutralized in 300 ml 0.5 M Tris-HCl, pH 8.0,0.15 M NaCl for the same duration. LOP was heated to 100°C for 5 min prior to hybridization. All hybridizations were carried out in Seal-a-Meal bags for 16-20 h in 5~ SSPE (1X SSPE = 180 mM NaCl, 10 mM sodium phosphate, and 1 mM EDTA, pH 8.1), 0.1% SDS, 10 fig/ml sonicated and denatured Escherichia coli DNA, and 1 X lo6 cpm/ ml labeled probe at 50°C for COP, and 65°C for LOP. After the hybridization the gel was washed in 6X SSC at room temperature for 2-3 h changing the washing solution after every 30 min, and washed gels were then exposed to X-ray film (Kodak X-AR) with or without intensifying screens (DuPont Lighting Plus) for 16-20 h depending upon the signal intensity. After the preliminary autoradiograms were obtained, the gels were given stringent washes in 6X SSC at 55-65°C for 25-35 min and autoradiograms were recorded. Usually, LOP hybridized filter withstood very high-stringent washes and thus these gels were washed in 3~ SSC and finally in 1.5X SSC and exposed without intensifying screens. The gels and filters hybridized with COP were subjected to similar treatment and autoradiograms were obtained. Similar results were obtained using Southern blot hybridization (18). RESULTS

Polyacrylamide gel electrophoresis of the ligation reaction demonstrated a heterogeneous population of various oligomers each different from one another with respect to their lengths from monomer (16 nucleotides long) to more than 15 units (240 nucleotides) (Figs. 1A

23.1 -

FIG. 2. (Left) Autoradiogram of the Hinfl-digested DNA from unrelated individuals (l-5) hybridized with COP 3315-16.1. Note the locus-specific nonpolymorphic 1.3-kb bands in all the individuals. (Right) The same gel hybridized with ligated oligonucleotide probe (LOP) and washed under very high stringency conditions. Note the several highly polymorphic bands in the range of 3-23 kb. For details, please see Materials and Methods.

and 1B). Because the ligation substrates were mixtures of more than one sequence, the products are also a heterogeneous population of sequence complexities. Human DNA samples were obtained from five different individuals, digested with Hi&, and subjected to agarose gel electrophoresis and the DNA was denatured and the gel was dried prior to hybridization. Hybridization of the gel with the 16-nucleotide-long conventional oligonucleotide probe shows a highly polymorphic pattern under nonstringent conditions, producing several resolvable bands in the range of 3 to 21 kb (not shown). However, the same probe produces a locus-specific, nonpolymorphic pattern when the gel is washed for 5 min at 55°C in 6X SSC. The nonpolymorphic single band (Fig. 2, left) of about 1.3 kb is found to be present in all the individuals studied thus far and represents the locus encoding the 33.15 cloned DNA isolated and sequenced by Jeffreys et al. (6). Unlike COP, hybridization with LOP gives a highly polymorphic pattern under both low-stringency and high-stringency wash conditions. The hybridization pattern is highly stable with LOP, and multiple resolvable polymorphic bands are seen in the range of 323 kb (Fig. 2, right). Fragments smaller than 3 kb are apparently nonpolymorphic. The patterns obtained are highly reproducible when the same DNA samples are compared on different gels. DISCUSSION

Ligation of oligonucleotides which represent a single repeat unit of a short tandemly repeated sequence pro-

ENZYMATIC

SYNTHESIS

vides an approach to obtain a long polymeric DNA probe which is totally synthetic. The advantage of this approach is that one can produce a probe without having to obtain it in a stable cloned form. Stability of minisatellite sequences in the cloned form has been described as a problem by Wong and co-workers (19). Furthermore, radiolabeling of the probe is accomplished by a simple kinase reaction performed on the starting monomeric oligonucleotide. Obviously, probes which are based on hypothesis rather than observation can be constructed to test the existence of sequences in a particular DNA. In this study, the 16-nucleotide-long COP produces a nonpolymorphic, locus-specific band under high-stringency wash conditions. This probe is not useful for DNA fingerprint analysis or for monitoring the inheritance pattern among family members. We have described this locus in detail in a previous study (17), where we used a 32-nucleotide-long probe (two repeat units) in addition to a 16-nucleotide-long probe as reported here. In both hybridization studies, the pattern under high-stringency wash conditions demonstrated a nonpolymorphic band of 1.3 kb. This probe, however, gives a very different hybridization profile when it is ligated into a polymeric probe. The pattern is consistently polymorphic and the hybridization is highly stable. We have analyzed DNA from several unrelated individuals and found that with LOP no two individuals are alike. Thus, the LOP behaves like a true DNA fingerprint probe. Synthesis of oligonucleotide sequences longer than 100 bases is difficult, particularly in the purification step, due, in part, to low overall yields. However, by the current approach reasonably large fragments can be obtained. We have been able to generate more than 500nucleotide-long probes by enzymatic ligation (data not shown). It may be possible to construct even longer probes by altering the reaction conditions. Because only one of the two oligonucleotides used in the ligation reaction is phosphorylated, the probe produced is effectively single stranded, since the short complementary strand cannot hybridize under the conditions used for LOP hybridization.

OF

DNA

283

PROBES

In view of these observations, we feel that this approach is yet an additional tool toward the understanding of the human genome by monitoring sequence polymorphism. ACKNOWLEDGMENTS This work was supported by Grant DCB-8515365 Science Foundation (R.B.W.). R.B.W. is a member ter of the City of Hope (NIH CA33672).

from the National of the Cancer Cen-

REFERENCES 1. Schaefer, R., Ah, S., and Eppien, J. T. (1986) Chromosoma 93, 502-510. 2. Martinex-Zapater, J. M., Estelle, M. A., and Sommerville, C. R. (1986) Mol. Gen. Genet. 204,417-423. 3. Bell, G. I., Selby, J., and Rutter, W. J. (1982) Nature fL.ondon,J

305,31-35. 4. Proudfoot, N. J., Gel, A., and Maniatis, 563.

T. (1982)

CeU 31,553-

5. Nakamura, Y., Leppert, M., O’Connell, P., Wolff, R., Holm, T., Culver, M., Martin, C., Fujimoto, E., Hoff, M., Kumlin, E., and White, R. (1987) Science 236,1616-1622. 6. Jeffreys, A. J., Wilson, V., and Thein, S. L. (1985) Nature (Lxxdon) 314,67-73. 7. White, R. (1985) Trends Genet. 1,177-X31. 8. Simmler, M. C., Johnsson, C., Petit, C., Rouyer, and Wissenbach, J. (1987) EMBO J. 6,963-969. 9. Ali, S., Mueller, C. R., and Epplen, J. T. (1986)

F., Vergnaud,

G.,

Hum.

74,

Genet.

239-245. 10. Yam, P., Petz, L. D., Ali, S., Stock, D., Amer. J. Hum. Genet. 41,867-881. 11. Jeffreys, A. J., Wilson, V., Thein, S. Ponder, B. A. J. (1986) Amer. J. Hum. 12. Jeffreys, A. J., Brookfield, J. F. Y., and ture (London) 317.818-819. 13. Gill,

P., Jeffreys,

A. J., and Werrett,

318,577-579. 14. Gill, P., Lygo, J. E., Fowler, phoresis

and Wallace,

R. B. (1987)

L., Weatherall, D. J., and Genet. 39,11-24. Semeonoff, R. (1985b) Na-

D. J. (1985)

S. J., and Werrett,

Nature

D. J. (1987)

(London) Electra-

8,36-44.

15. Hill, A. V. S., and Jeffreys, A. J. (1985) Lancet 1,1394-1395. 16. Georges, M., Lequarre, A.-S., Castelli, M., Hanset, R., and Vassart, G. (1988) Cytogenet. Cell Genet. 47,127-131. 17. Ali, S., and Wallace, R. B. (1988) Nucleic Acids Res. 16, 8487-

8496. 18. Southern, E. M. (1975) J. Mol. Biol. 98,503-517. 19. Wong, Z., Wilson, V., Jeffreys, A. J., and Thein, Nucleic Acids Res. 14,4606-4616.

S. L. (1986)