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Direct sequencing of variable HLA gene segments after in vitro amplification and allele separation by temperature-gradient gel electrophoresis
Journal of Immunological Methods, 142 (1991) 251-256 © 1991 Elsevier Science Publishers B.V. All rights reserved 0022-1759/91/$03.50 ADONIS 002217599100282X
251
JIM06057
Direct sequencing of variable HLA gene segments after in vitro amplification and allele separation by temperature-gradient gel electrophoresis Christian G. M e y e r 1, Egbert Tannich 1, Jutta H a r d e r s 2, Karsten H e n c o 2 and R o l f D. H o r s t m a n n 1 1 Bernhard Nocht Institute for Tropical Medicine, 2000 Hamburg 36, F.R.G., and 2 Diagen GmbH, 4000 Diisseldorf F.R.G.
(Received 12 February 1991, revised received 20 May 1991, accepted 21 May 1991)
Previously unrecognized variants of human leukocyte antigens (HLA) are currently being analyzed by in vitro amplification and sequencing of the variable gene segments. In heterozygous individuals, molecular cloning is required to separate the two concomitantly amplified haplotypic gene segments. A method is presented which facilitates the procedure of separating the two haplotypic gene segments by using a temperature-gradient gel electrophoresis (TGGE). The procedure comprises PCR amplification of the variable HLA gene segments, allele separation by TGGE, re-amplification of each of the separated allelic segments, and direct DNA sequencing using the PCR primers. Key words." Human leukocyte antigen; Major histocompatibility complex; Temperature-gradient gel electrophoresis; Direct sequencing
Introduction
In recent years structural analyses of HLA polymorphisms have been refined by sequencing of the variable gene segments. This resulted in the recognition of additional allelic variants and in the resolution of disease associations (Scharf et al., 1988; Begovich et al., 1989; Erlich et al., 1990). Application of the polymerase chain reaction (PCR) using locus-specific primers allowed
Correspondence to: C.G. Meyer, Bernhard Nocht Institute for Tropical Medicine, 2000 Hamburg 36, F.R.G. Abbreviations." bp, base pair; EDTA, ethylene-diaminetetraacetic acid; HLA, human leukocyte antigen; PCR, polymerase chain reaction; TGGE, temperature-gradient gel electrophoresis.
the isolation and amplification of the variable gene segments in a one-step-procedure. In heterozygous individuals, however, locus-specific gene amplification yielded material consisting of a mixture of the two haplotypic gene segments. These used to be separated by molecular cloning procedures and subsequent sequencing of randomly selected clones whereby the probability of one allele being missed could only be reduced by increasing numbers of clones being sequenced. Here we present a procedure which uses temperature-gradient gel electrophoresis (TGGE) for the separation of the two haplotypic DNA segments. After successive PCR amplification, allele separation by TGGE, and re-amplification of each of the allelic segments, the variable HLA gene segments are analyzed by direct DNA sequencing
252 using the PCR primers. The procedure facilitates the identification of previously unrecognized HLA alleles avoiding recombinant technology. It is presented on the example of the variable gene segment of the human class II DPA1 locus.
Materials and methods
Isolation of genomic DNA Human peripheral blood leukocytes were isolated as described (Sambrook et al., 1989). Approximately 5 X 106 cells were pelleted and resuspended in a 10 mM Tris-HCl buffer, pH 7.6, containing 10 mM EDTA, 50 mM NaC1, and 0.2% SDS. DNA was released by treatment with proteinase K (0.3 mg/ml) at 56°C for 3 h, extracted once with phenol/chloroform 1:1 (v/v), once with chloroform 1 : 1 (v/v), and precipitated with ethanol (2.5 vols. in 0.3 M sodium acetate). The resulting DNA pellet was resuspended in 50 /~1 TE buffer (10 mM Tris pH 7.6, 1 mM EDTA) and dialyzed against 2 x 2 ml TE buffer (Centricon 100, Amicon) to remove salt and traces of SDS.
Amplification procedures Two oligonucleotide primers (Bugawan et al., 1988) GH 98 (5'-CGCGGATCCTGTGTCAACTTATGCCGC-3') and GH 99 (5'-CTGGCTGCAGTGTGGTTGGAACGC-3') were synthesized on an Applied Biosystems DNA synthesizer. PCR was performed as described (Saiki et al., 1988). Briefly, 0.5-1.0 /xg of genomic target DNA, 100 pmol of each primer, and 2.5 U of Taq polymerase (Cetus Corp.) were applied in a 100 tzl reaction volume containing a 10 mM Tris-HC1 buffer, pH 8.3, 50 mM KCI, 1.5 mM MgCI 2, 0.01% gelatin, and 0.2 mM deoxynucleotide triphosphates. Samples were amplified for 35 cycles in an automated Thermal Cycler (PerkinElmer-Cetus). Each cycle consisted of 0.5 min of denaturation at 94 ° C, 1.0 min of annealing at 55°C, and 2.0 min of extension at 72 °C. To ascertain completion of DNA synthesis, the last extension step was prolonged to 10 min. Amplified DNA was purified by preparative electrophoresis in low melting point agarose (Sigma).
Temperature-gradient gel electrophoresis (TGGE) The temperature-gradient gel electrophoreses (TGGE, Diagen, Dfisseldorf, F.R.G.) (Riesner et al., 1989) were performed in horizontal 7.5% polyacrylamide gels (30/0.5, acrylamide/bisacrylamide) prepared in 0.1 x TBE, pH 8.3, (8.9 mM Tris base, 8.9 mM boric acid, and 0.25 mM Na 2 EDTA) containing 4 M urea. Electrophoretic running buffer was 0.1 x TBE, and DNA was dissolved in 0.1 x TBE containing bromophenol blue and xylene cyanol FF dye. Electrophoreses were started at 12 °C with 300 V for 30 min and continued under temperature-gradient conditions for approximately 2.5 h. The gradients were applied either in parallel or perpendicularly, i.e., in a right angle, to the electric field. In both cases, temperature gradients were used from 17 °C to 60 o C. In case of gradients parallel to the electric field, 300 V were continued throughout the runs; with temperature gradients applied perpendicularly, the voltage was increased to 500 V.
Re-amplification and DNA sequencing After electrophoretic separation, DNA bands were identified by silver staining. After fixation in 10% ethanol/0.5% acetic acid for 3 rain, the gels were stained in 0.1% silver nitrate for 10 min, washed twice in destilled water, developed with 1.5% NaOH/0.01% NaBH4/0.15% formaldehyde for 20 min, and fixed in 0.75% NazCO 3 for 10 rain with gentle shaking during each step. DNA containing gel slices were cut out, and DNA was eluted into 200/zl of a 10 mM Tris-HCl buffer, pH 7.4, containing 1 mM EDTA (TE buffer) by incubation at 37 ° C for 4 h or overnight under vigorous shaking. The eluate was subjected to ethanol precipitation (2.5 vols. ethanol in 0.3 M sodium acetate) and vacuum drying. The precipitated DNA was resuspended in 15 /zl H20, and 3 #1 were applied to another PCR amplification (re-amplification) using the same primers and amplification protocol as described above. Before sequencing, the products of the re-amplification were purified by preparative gel electrophoresis in low melting point agarose. Both strands were sequenced following the procedure of Casanova et al. (1990) using the PCR primers GH 98 and GH 99. To further reduce rapid
253
DPAI*0101
s t r a n d reassociation, the s e q u e n c i n g r e a c t i o n mixture was m a d e to c o n t a i n 5 % N P 40.
M
÷
DPA1*0201
bp
Results
First amplification G e n o m i c D N A was isolated from leukocytes of 1 - 2 ml blood. A p p r o x i m a t e l y 0 . 5 - 1 . 0 /~g D N A was subjected to P C R amplification using the p r i m e r s G H 98 a n d G H 99 which were derived from the 5' a n d 3' e n d s of the second exon of the h u m a n D P A 1 gene, respectively (Fig. 1) (Bugaw a n et al., 1988). T h u s , a 243 bp s e g m e n t was amplified which c o m p r i s e d two o u t of the t h r e e variable c o d o n s r e c o g n i z e d so far o n the D P A 1 locus (Figl 2). T h e P C R c o n d i t i o n s were c h o s e n to yield h o m o g e n e o u s d o u b l e - s t r a n d e d D N A . T h e amplified D N A was purified by p r e p a r a t i v e gel electrophoresis, p h e n o l extraction, a n d e t h a n o l precipitation.
267 234
Fig. 2. Agarose gel electrophoresis of DPA1 DNA amplified' by PCR. Genomic DNA (0.5-1 /xg) of peripheral blood leukocytes was amplified for 35 cycles using the DPA1 primers GH 98 and GH 99 (Bugawan et al., 1988) (see Fig. 1). 10/xl of the 100 p~l PCR reaction volume were resolved in 1.8% agarose. Left lane: molecular weight markers (pBR322-DNA restricted with endonuclease HAE III, Boehringer Mannheim, F.R.G.); right lane: the PCR amplified 243 bp HLA-DPA1 product. Segment sizes are indicated in base pairs (bp).
Temperature-gradient gel electrophoresis I T w o a l t e r n a t i v e e l e c t r o p h o r e t i c r e g i m e n s were applied. First, the t e m p e r a t u r e g r a d i e n t was established p e r p e n d i c u l a r l y , i.e., in a right angle to the e l e c t r o p h o r e t i c r u n n i n g direction. T h e e n t i r e
DPAI*0101 DPAI'0201 DPAI*0101 DPAI*0201
~ H 98 nu ATC AAG GCG GAC CAT GTG TCA ACT TAT GCC GCG TTT GTA CAG ACG nu ............................................................... aa I K A D H V S T Y A A F V Q T aa . . . . . . . . . . . . . . . . .
CAT
AGA
CCA
ACA
GGG
GAG
H
R
P
T
G
E
ATG TTC TAT GTG GAT CTG GAC AAG AAG GAG CA .................................. M P Y V D L D K K E Q . . . . . . . . . . .
ACC
GTC
T
V
TGG CAT CTG GAG GAG TTT GGC CAA GCC TTT TCC TTT GAG GCT CAG GGC GGG CTG GCT ...................... G ........................................ W H L E E F G Q A F S F E A Q G G L A . . . . . . . . R. . . . . . . . . . . .
AAC
ATT
N
I
GCT ATA TTG AAC AAC AAC TTG AAT ACC TTG ATC CAG CGT TCC ......................................................... A I L N N N L N T L I Q R S . . . . . . . . . . . . . . . .
Ace AAC G ..... T N A
TTT ATG TTT GAA TTT GAT GAA GAT ........................... F M T E F D E D . . . . . . . . .
GAG E
.
.
.
.
AAC
CAC
ACT
CAG
GCC
N
H
T
Q
A
.
. G H 99
Fig. 1. Alignments of the nucleotide (nu) and deduced amino acid (aa) sequences of the second exon of DPAI*0101 and DPAI*0201. Amino acid sequences are shown in the single-letter code. For DPAI* 0201, only those nucleotides and amino acids are shown which differ from those of DPAI*0101; identical residues are indicated by dashes. The position of the primers GH 98 and GH 99 used for PCR amplification and sequencing are shown by solid lines. The PCR amplification yielded 243 bp DNA segments comprising two out of the three variable codons.
254
DPAl’ Ol+o’
DPAl* 0101
DPAl* 0201
?I%*
v
,
17oc
b60°C
Fig. 3. Perpendicular TGGE for the separation of the two allelic segments of the purified HLA DPAl amplification product. The gel consisted of 7.5% polyacrylamide and contained 4 M urea. Approximately 3 pg of double stranded DNA were applied. After ‘pre-electrophoresis’ at 300 V and 12 o C for 30 min, a temperature gradient from 17 o C (left) to 60 OC (right) was applied perpendicularly, i.e., in a right angle to the electrophoretic running direction, and electrophoresis was continued at 500 V for 2.5 h. Silver staining revealed the two DNA species as two distinct bands forming in the gel region where the temperature was such that the two HLA DPAI allelic segments exhibited different melting properties.
amplified material of approximately 3 pg was loaded onto the entire breadth of the gel. After silver staining, the presence of two DNA species representing the allelic variants was evident from two distinct bands forming in a temperature range where the melting properties of the two allelic gene segments were different (Fig. 3).
Temperature-gradient
gel electrophoresis II
Alternatively, the temperature gradient was applied in parallel to the electric field. l/10 of the amplification product was applied corresponding to approximately 0.3 pg DNA. The two allelic gene segments were being separated while the DNA was migrating through the circumscribed part of the temperature gradient in which the two allelic gene segments have different melting properties (Fig. 4). Hence it was important not to terminate the electrophoresis before this particular part of the gradient was reached by the migrating DNA.
efier l%R
2. Pm
Fig, 4. Parallel TGGE for separation of the two allelic segments of the purified HLA DPAl amplification product. A temperature gradient between 17 o C (top) and 60 o C (bottom) was applied in parallel to the electrophoretic running direction. Lane I: electrophoretic separation of the amphfication product. Lanes 2 and 3: reelectrophoresis of the two separated HLA DPAl allelic segments after reamplification.
255
Second amplification and DNA sequencing PCR re-amplification was performed using DNA eluted from the silver stained TGGE gels as a template. By direct sequencing of the reamplified PCR products, the presence of the two DNA species corresponding to the HLA DPA1 alleles DPAI*0101 and DPAI*0201 was demonstrated.
Discussion
The technique of HLA-typing by oligonucleotide probing has recently been developed and is considered to greatly enhance the accuracy of the typing procedure. The method implies PCR amplification of the variable gene segments. This typing procedure can be supplemented by temperature-gradient gel electrophoresis (TGGE). As previously described, double stranded DNA molecules differing in a few nucleotides or even one single base position can be separated using denaturing gels (l_~rman et al., 1984; Riesner et al., 1989). TGGE may have two applications, namely (i) to support evidence for homozygosity, and (ii) to separate the two allelic segments derived from a heterozygous individual for DNA sequencing. The first application still requires experimental evaluation. To support evidence for homozygosity, TGGE would be useful as an analytical method. One might suggest that for this purpose the perpendicular gradient is superior to the parallel one because the shape of the DNA band across the temperature range visualizes that partial and complete melting has occurred in distinct parts of the gradient. Thus, perpendicular TGGE can convincingly illustrate identity or non-identity of the DNA segments applied. In contrast, electrophoresis with a parallel gradient shows the final product of the run only, and a lack of separation may indicate identity but may also be due to technical mistakes such as premature termination of the run or other technical problems. Once a system is established, however, parallel gradients have the advantage of allowing multiple sample analyses. he second application is presented in this report. It uses TGGE as a preparative method. In
the case of heterozygous individuals, PCR amplification using HLA locus-specific primers yields two allelic segments identical in length and highly similar in sequence. Before sequencing, the two segments have to be separated. It may be assumed that, once fully established, HLA typing by oligonucleotide probing may widely replace the sequencing procedure. However, sequencing may still be required (i) if DNA typing shows a hybridization pattern not attributable to any of the known alleles, or (ii) if circumstantial evidence such as a suspected disease association suggests a polymorphism which is not revealed by DNA typing. To prepare the PCR product of a heterozygous individual for sequencing, several strategies have been proposed including allele-specific amplification, direct sequencing of one or each of the allelic segments by using allele-specific primers, and separation of the allelic segments by denaturing gradient gel electrophoresis prior to sequencing. However, the first two approaches require precise sequence information on both allelic variants which are usually not available in instances where DNA typing fails, and the latter approach has not been elaborated in detail (Gyllensten, 1989). Currently, the two allelic segments are being separated by molecular cloning: clones are selected at random, and usually one has to sequence a number of them in order not to miss one of the two alleles. In contrast to the random approach, preparative temperature-gradient gel electrophoresis allows the positive distinction and recovery of the two allelic gene segments, at the same time avoiding recombinant technology. In general, the use of the parallel temperature gradient may be sufficient; it has the advantage that several samples can be run simultaneously. Perpendicular gradient gels may be applied only if the result obtained with parallel conditions is ambiguous. We have shown that DNA eluted from silver-stained TGGE gels can be reliably reamplified by PCR. The sequencing protocol for double stranded PCR products (Casanova et al., 1990) was slightly modified by including a nonionic detergent in the reaction mixture to further reduce reassociation of the single-stranded DNA. Thus asymmetric PCR may be avoided. In addition to the application presented here,
256 the m e t h o d of t e m p e r a t u r e - g r a d i e n t gel electrophoresis offers a b r o a d s p e c t r u m of o t h e r applications in research ( R i e s n e r et al., 1991). Besides the d e t e c t i o n a n d p r e p a r a t i o n of polymorphic m u t a n t s in genetics a n d in the analysis of infectious diseases, T G G E is a reliable a n d easyt o - p e r f o r m t e c h n i q u e to achieve q u a n t i t a t i v e P C R for the d e t e r m i n a t i o n of exact copy n u m b e r s of specific genes or transcripts in biological samples ( H e n c o a n d Heibey, 1990). T G G E is n o t limited, however, to the analysis of p u r e nucleic acids. It may also be used for the c h a r a c t e r i z a t i o n of proteins or p r o t e i n / n u c l e i c acid complexes such as m u l t i - f a c t o r complexes in g e n e r e g u l a t i o n (Wag e n h f f e r et al., 1988).
References Begovich, A.B., Bugawan, T.L., Nepom, B.S., Klitz, W., Nepom, G.T. and Erlich, H.A. (1990) A specific HLA-DPfl allele is associated with pauciarticular juvenile rheumatoid arthritis but not adult rheumatoid arthritis. Proc. Natl. Acad. Sci. U.S.A. 86, 9489. Bugawan, T.L., Horn, G.T., Long, C.M., Mickelson, E., Hansen, J.A., Ferrara, G.B., Angelini, G. and Erlich, H.A. (1988) Analysis of HLA-DP allelic sequence polymorphism using the in vitro enzymatic DNA amplification of DPA and DPB loci. J. Immunol. 141, 4024. Casanova, J.-L., Pannetier, C., Jaulin, C. and Kourilsky, P. (1990) Optimal conditions for directly sequencing doublestranded PCR products with sequenase. Nucleic Acids Res. 18, 4028. Erlich, H.A., Bugawan, T.L., Scharf, S., Nepom, G.T., Tait, B. and Griffith, R.L. (1990) HLA-DQ/3 sequence polymor-
phism and genetic susceptibility to IDDM. Diabetes 39, 96. Gyllensten, U. (1989) Direct sequencing of in vitro amplified DNA. In: H.A. Erlich (Ed.), PCR Technology. Stockton Press, New York, p. 45. Henco, K. and Heibey, M. (1990) Quantitative PCR - the determination of template copy numbers by temperaturegradient gel electrophoresis. Nucleic Acids Res. 18, 6733. Lerman, L.S., Fischer, S.G., Hurley, I., Silverstein, K. and Lumelsky, N. (1984) Sequence-determinated DNA separations. Ann. Rev. Biophys. Bioeng. 13, 399. Riesner, D., Steger, G., Zimmat, R., Owens, R.A., Wagenh6fer, M., Hillen, W., Vollbach, S. and Henco, K. (1989) Temperature-gradient gel electrophoresis of nucleic acids: analysis of conformational transitions, sequence variations, and protein-nucleic acid interactions. Electrophoresis 10, 377. Riesner, D., Henco, K. and Steger, G. (1991) Temperaturegradient gel electrophoresis: a method for the confirmational transitions and mutations in nucleic acids and proteins. In: A. Crambach (Ed.), Advances in Electrophoresis, Vol. 4. VCH, Weinheim. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487. Sarnbrook, J., Fritsch, E.F. and Maniatis, T. (1989) In: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York. Scharf, S.J., Friedman, A., Brautbar, C., Szafer, F., Steinmann, L., Horn, G., Gyllensten, U. and Erlich, H.A. (1988) HLA class II allelic variation and susceptibility to pemphigus vulgaris. Proc. Natl. Acad. Sci. U.S.A. 85, 3508. Wagenhffer, M., Hansen, D. and Hillen, W. (1988) Thermal denaturation of engineered tet repressor protein and their complexes with tet operator and tetracycline studied by temperature-gradient gel electrophoresis. Anal. Biochem. 175, 422.
251
JIM06057
Direct sequencing of variable HLA gene segments after in vitro amplification and allele separation by temperature-gradient gel electrophoresis Christian G. M e y e r 1, Egbert Tannich 1, Jutta H a r d e r s 2, Karsten H e n c o 2 and R o l f D. H o r s t m a n n 1 1 Bernhard Nocht Institute for Tropical Medicine, 2000 Hamburg 36, F.R.G., and 2 Diagen GmbH, 4000 Diisseldorf F.R.G.
(Received 12 February 1991, revised received 20 May 1991, accepted 21 May 1991)
Previously unrecognized variants of human leukocyte antigens (HLA) are currently being analyzed by in vitro amplification and sequencing of the variable gene segments. In heterozygous individuals, molecular cloning is required to separate the two concomitantly amplified haplotypic gene segments. A method is presented which facilitates the procedure of separating the two haplotypic gene segments by using a temperature-gradient gel electrophoresis (TGGE). The procedure comprises PCR amplification of the variable HLA gene segments, allele separation by TGGE, re-amplification of each of the separated allelic segments, and direct DNA sequencing using the PCR primers. Key words." Human leukocyte antigen; Major histocompatibility complex; Temperature-gradient gel electrophoresis; Direct sequencing
Introduction
In recent years structural analyses of HLA polymorphisms have been refined by sequencing of the variable gene segments. This resulted in the recognition of additional allelic variants and in the resolution of disease associations (Scharf et al., 1988; Begovich et al., 1989; Erlich et al., 1990). Application of the polymerase chain reaction (PCR) using locus-specific primers allowed
Correspondence to: C.G. Meyer, Bernhard Nocht Institute for Tropical Medicine, 2000 Hamburg 36, F.R.G. Abbreviations." bp, base pair; EDTA, ethylene-diaminetetraacetic acid; HLA, human leukocyte antigen; PCR, polymerase chain reaction; TGGE, temperature-gradient gel electrophoresis.
the isolation and amplification of the variable gene segments in a one-step-procedure. In heterozygous individuals, however, locus-specific gene amplification yielded material consisting of a mixture of the two haplotypic gene segments. These used to be separated by molecular cloning procedures and subsequent sequencing of randomly selected clones whereby the probability of one allele being missed could only be reduced by increasing numbers of clones being sequenced. Here we present a procedure which uses temperature-gradient gel electrophoresis (TGGE) for the separation of the two haplotypic DNA segments. After successive PCR amplification, allele separation by TGGE, and re-amplification of each of the allelic segments, the variable HLA gene segments are analyzed by direct DNA sequencing
252 using the PCR primers. The procedure facilitates the identification of previously unrecognized HLA alleles avoiding recombinant technology. It is presented on the example of the variable gene segment of the human class II DPA1 locus.
Materials and methods
Isolation of genomic DNA Human peripheral blood leukocytes were isolated as described (Sambrook et al., 1989). Approximately 5 X 106 cells were pelleted and resuspended in a 10 mM Tris-HCl buffer, pH 7.6, containing 10 mM EDTA, 50 mM NaC1, and 0.2% SDS. DNA was released by treatment with proteinase K (0.3 mg/ml) at 56°C for 3 h, extracted once with phenol/chloroform 1:1 (v/v), once with chloroform 1 : 1 (v/v), and precipitated with ethanol (2.5 vols. in 0.3 M sodium acetate). The resulting DNA pellet was resuspended in 50 /~1 TE buffer (10 mM Tris pH 7.6, 1 mM EDTA) and dialyzed against 2 x 2 ml TE buffer (Centricon 100, Amicon) to remove salt and traces of SDS.
Amplification procedures Two oligonucleotide primers (Bugawan et al., 1988) GH 98 (5'-CGCGGATCCTGTGTCAACTTATGCCGC-3') and GH 99 (5'-CTGGCTGCAGTGTGGTTGGAACGC-3') were synthesized on an Applied Biosystems DNA synthesizer. PCR was performed as described (Saiki et al., 1988). Briefly, 0.5-1.0 /xg of genomic target DNA, 100 pmol of each primer, and 2.5 U of Taq polymerase (Cetus Corp.) were applied in a 100 tzl reaction volume containing a 10 mM Tris-HC1 buffer, pH 8.3, 50 mM KCI, 1.5 mM MgCI 2, 0.01% gelatin, and 0.2 mM deoxynucleotide triphosphates. Samples were amplified for 35 cycles in an automated Thermal Cycler (PerkinElmer-Cetus). Each cycle consisted of 0.5 min of denaturation at 94 ° C, 1.0 min of annealing at 55°C, and 2.0 min of extension at 72 °C. To ascertain completion of DNA synthesis, the last extension step was prolonged to 10 min. Amplified DNA was purified by preparative electrophoresis in low melting point agarose (Sigma).
Temperature-gradient gel electrophoresis (TGGE) The temperature-gradient gel electrophoreses (TGGE, Diagen, Dfisseldorf, F.R.G.) (Riesner et al., 1989) were performed in horizontal 7.5% polyacrylamide gels (30/0.5, acrylamide/bisacrylamide) prepared in 0.1 x TBE, pH 8.3, (8.9 mM Tris base, 8.9 mM boric acid, and 0.25 mM Na 2 EDTA) containing 4 M urea. Electrophoretic running buffer was 0.1 x TBE, and DNA was dissolved in 0.1 x TBE containing bromophenol blue and xylene cyanol FF dye. Electrophoreses were started at 12 °C with 300 V for 30 min and continued under temperature-gradient conditions for approximately 2.5 h. The gradients were applied either in parallel or perpendicularly, i.e., in a right angle, to the electric field. In both cases, temperature gradients were used from 17 °C to 60 o C. In case of gradients parallel to the electric field, 300 V were continued throughout the runs; with temperature gradients applied perpendicularly, the voltage was increased to 500 V.
Re-amplification and DNA sequencing After electrophoretic separation, DNA bands were identified by silver staining. After fixation in 10% ethanol/0.5% acetic acid for 3 rain, the gels were stained in 0.1% silver nitrate for 10 min, washed twice in destilled water, developed with 1.5% NaOH/0.01% NaBH4/0.15% formaldehyde for 20 min, and fixed in 0.75% NazCO 3 for 10 rain with gentle shaking during each step. DNA containing gel slices were cut out, and DNA was eluted into 200/zl of a 10 mM Tris-HCl buffer, pH 7.4, containing 1 mM EDTA (TE buffer) by incubation at 37 ° C for 4 h or overnight under vigorous shaking. The eluate was subjected to ethanol precipitation (2.5 vols. ethanol in 0.3 M sodium acetate) and vacuum drying. The precipitated DNA was resuspended in 15 /zl H20, and 3 #1 were applied to another PCR amplification (re-amplification) using the same primers and amplification protocol as described above. Before sequencing, the products of the re-amplification were purified by preparative gel electrophoresis in low melting point agarose. Both strands were sequenced following the procedure of Casanova et al. (1990) using the PCR primers GH 98 and GH 99. To further reduce rapid
253
DPAI*0101
s t r a n d reassociation, the s e q u e n c i n g r e a c t i o n mixture was m a d e to c o n t a i n 5 % N P 40.
M
÷
DPA1*0201
bp
Results
First amplification G e n o m i c D N A was isolated from leukocytes of 1 - 2 ml blood. A p p r o x i m a t e l y 0 . 5 - 1 . 0 /~g D N A was subjected to P C R amplification using the p r i m e r s G H 98 a n d G H 99 which were derived from the 5' a n d 3' e n d s of the second exon of the h u m a n D P A 1 gene, respectively (Fig. 1) (Bugaw a n et al., 1988). T h u s , a 243 bp s e g m e n t was amplified which c o m p r i s e d two o u t of the t h r e e variable c o d o n s r e c o g n i z e d so far o n the D P A 1 locus (Figl 2). T h e P C R c o n d i t i o n s were c h o s e n to yield h o m o g e n e o u s d o u b l e - s t r a n d e d D N A . T h e amplified D N A was purified by p r e p a r a t i v e gel electrophoresis, p h e n o l extraction, a n d e t h a n o l precipitation.
267 234
Fig. 2. Agarose gel electrophoresis of DPA1 DNA amplified' by PCR. Genomic DNA (0.5-1 /xg) of peripheral blood leukocytes was amplified for 35 cycles using the DPA1 primers GH 98 and GH 99 (Bugawan et al., 1988) (see Fig. 1). 10/xl of the 100 p~l PCR reaction volume were resolved in 1.8% agarose. Left lane: molecular weight markers (pBR322-DNA restricted with endonuclease HAE III, Boehringer Mannheim, F.R.G.); right lane: the PCR amplified 243 bp HLA-DPA1 product. Segment sizes are indicated in base pairs (bp).
Temperature-gradient gel electrophoresis I T w o a l t e r n a t i v e e l e c t r o p h o r e t i c r e g i m e n s were applied. First, the t e m p e r a t u r e g r a d i e n t was established p e r p e n d i c u l a r l y , i.e., in a right angle to the e l e c t r o p h o r e t i c r u n n i n g direction. T h e e n t i r e
DPAI*0101 DPAI'0201 DPAI*0101 DPAI*0201
~ H 98 nu ATC AAG GCG GAC CAT GTG TCA ACT TAT GCC GCG TTT GTA CAG ACG nu ............................................................... aa I K A D H V S T Y A A F V Q T aa . . . . . . . . . . . . . . . . .
CAT
AGA
CCA
ACA
GGG
GAG
H
R
P
T
G
E
ATG TTC TAT GTG GAT CTG GAC AAG AAG GAG CA .................................. M P Y V D L D K K E Q . . . . . . . . . . .
ACC
GTC
T
V
TGG CAT CTG GAG GAG TTT GGC CAA GCC TTT TCC TTT GAG GCT CAG GGC GGG CTG GCT ...................... G ........................................ W H L E E F G Q A F S F E A Q G G L A . . . . . . . . R. . . . . . . . . . . .
AAC
ATT
N
I
GCT ATA TTG AAC AAC AAC TTG AAT ACC TTG ATC CAG CGT TCC ......................................................... A I L N N N L N T L I Q R S . . . . . . . . . . . . . . . .
Ace AAC G ..... T N A
TTT ATG TTT GAA TTT GAT GAA GAT ........................... F M T E F D E D . . . . . . . . .
GAG E
.
.
.
.
AAC
CAC
ACT
CAG
GCC
N
H
T
Q
A
.
. G H 99
Fig. 1. Alignments of the nucleotide (nu) and deduced amino acid (aa) sequences of the second exon of DPAI*0101 and DPAI*0201. Amino acid sequences are shown in the single-letter code. For DPAI* 0201, only those nucleotides and amino acids are shown which differ from those of DPAI*0101; identical residues are indicated by dashes. The position of the primers GH 98 and GH 99 used for PCR amplification and sequencing are shown by solid lines. The PCR amplification yielded 243 bp DNA segments comprising two out of the three variable codons.
254
DPAl’ Ol+o’
DPAl* 0101
DPAl* 0201
?I%*
v
,
17oc
b60°C
Fig. 3. Perpendicular TGGE for the separation of the two allelic segments of the purified HLA DPAl amplification product. The gel consisted of 7.5% polyacrylamide and contained 4 M urea. Approximately 3 pg of double stranded DNA were applied. After ‘pre-electrophoresis’ at 300 V and 12 o C for 30 min, a temperature gradient from 17 o C (left) to 60 OC (right) was applied perpendicularly, i.e., in a right angle to the electrophoretic running direction, and electrophoresis was continued at 500 V for 2.5 h. Silver staining revealed the two DNA species as two distinct bands forming in the gel region where the temperature was such that the two HLA DPAI allelic segments exhibited different melting properties.
amplified material of approximately 3 pg was loaded onto the entire breadth of the gel. After silver staining, the presence of two DNA species representing the allelic variants was evident from two distinct bands forming in a temperature range where the melting properties of the two allelic gene segments were different (Fig. 3).
Temperature-gradient
gel electrophoresis II
Alternatively, the temperature gradient was applied in parallel to the electric field. l/10 of the amplification product was applied corresponding to approximately 0.3 pg DNA. The two allelic gene segments were being separated while the DNA was migrating through the circumscribed part of the temperature gradient in which the two allelic gene segments have different melting properties (Fig. 4). Hence it was important not to terminate the electrophoresis before this particular part of the gradient was reached by the migrating DNA.
efier l%R
2. Pm
Fig, 4. Parallel TGGE for separation of the two allelic segments of the purified HLA DPAl amplification product. A temperature gradient between 17 o C (top) and 60 o C (bottom) was applied in parallel to the electrophoretic running direction. Lane I: electrophoretic separation of the amphfication product. Lanes 2 and 3: reelectrophoresis of the two separated HLA DPAl allelic segments after reamplification.
255
Second amplification and DNA sequencing PCR re-amplification was performed using DNA eluted from the silver stained TGGE gels as a template. By direct sequencing of the reamplified PCR products, the presence of the two DNA species corresponding to the HLA DPA1 alleles DPAI*0101 and DPAI*0201 was demonstrated.
Discussion
The technique of HLA-typing by oligonucleotide probing has recently been developed and is considered to greatly enhance the accuracy of the typing procedure. The method implies PCR amplification of the variable gene segments. This typing procedure can be supplemented by temperature-gradient gel electrophoresis (TGGE). As previously described, double stranded DNA molecules differing in a few nucleotides or even one single base position can be separated using denaturing gels (l_~rman et al., 1984; Riesner et al., 1989). TGGE may have two applications, namely (i) to support evidence for homozygosity, and (ii) to separate the two allelic segments derived from a heterozygous individual for DNA sequencing. The first application still requires experimental evaluation. To support evidence for homozygosity, TGGE would be useful as an analytical method. One might suggest that for this purpose the perpendicular gradient is superior to the parallel one because the shape of the DNA band across the temperature range visualizes that partial and complete melting has occurred in distinct parts of the gradient. Thus, perpendicular TGGE can convincingly illustrate identity or non-identity of the DNA segments applied. In contrast, electrophoresis with a parallel gradient shows the final product of the run only, and a lack of separation may indicate identity but may also be due to technical mistakes such as premature termination of the run or other technical problems. Once a system is established, however, parallel gradients have the advantage of allowing multiple sample analyses. he second application is presented in this report. It uses TGGE as a preparative method. In
the case of heterozygous individuals, PCR amplification using HLA locus-specific primers yields two allelic segments identical in length and highly similar in sequence. Before sequencing, the two segments have to be separated. It may be assumed that, once fully established, HLA typing by oligonucleotide probing may widely replace the sequencing procedure. However, sequencing may still be required (i) if DNA typing shows a hybridization pattern not attributable to any of the known alleles, or (ii) if circumstantial evidence such as a suspected disease association suggests a polymorphism which is not revealed by DNA typing. To prepare the PCR product of a heterozygous individual for sequencing, several strategies have been proposed including allele-specific amplification, direct sequencing of one or each of the allelic segments by using allele-specific primers, and separation of the allelic segments by denaturing gradient gel electrophoresis prior to sequencing. However, the first two approaches require precise sequence information on both allelic variants which are usually not available in instances where DNA typing fails, and the latter approach has not been elaborated in detail (Gyllensten, 1989). Currently, the two allelic segments are being separated by molecular cloning: clones are selected at random, and usually one has to sequence a number of them in order not to miss one of the two alleles. In contrast to the random approach, preparative temperature-gradient gel electrophoresis allows the positive distinction and recovery of the two allelic gene segments, at the same time avoiding recombinant technology. In general, the use of the parallel temperature gradient may be sufficient; it has the advantage that several samples can be run simultaneously. Perpendicular gradient gels may be applied only if the result obtained with parallel conditions is ambiguous. We have shown that DNA eluted from silver-stained TGGE gels can be reliably reamplified by PCR. The sequencing protocol for double stranded PCR products (Casanova et al., 1990) was slightly modified by including a nonionic detergent in the reaction mixture to further reduce reassociation of the single-stranded DNA. Thus asymmetric PCR may be avoided. In addition to the application presented here,
256 the m e t h o d of t e m p e r a t u r e - g r a d i e n t gel electrophoresis offers a b r o a d s p e c t r u m of o t h e r applications in research ( R i e s n e r et al., 1991). Besides the d e t e c t i o n a n d p r e p a r a t i o n of polymorphic m u t a n t s in genetics a n d in the analysis of infectious diseases, T G G E is a reliable a n d easyt o - p e r f o r m t e c h n i q u e to achieve q u a n t i t a t i v e P C R for the d e t e r m i n a t i o n of exact copy n u m b e r s of specific genes or transcripts in biological samples ( H e n c o a n d Heibey, 1990). T G G E is n o t limited, however, to the analysis of p u r e nucleic acids. It may also be used for the c h a r a c t e r i z a t i o n of proteins or p r o t e i n / n u c l e i c acid complexes such as m u l t i - f a c t o r complexes in g e n e r e g u l a t i o n (Wag e n h f f e r et al., 1988).
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