1146 Nucleic Acids Research, Vol. 20, No. 5
Rapid detection of sequence variations using polymers of specific oligonucleotides William A.Rudert and Massimo Trucco Division of Immunogenetics, Department of Pediatrics, University of Pittsburgh School of Medicine, Rangos Research Center, Children's Hospital of Pittsburgh, Pittsburgh, PA 15213, USA Submitted January 23, 1992 Hybridization with sequence-specific oligonucleotides has become the preferred method for detecting known variations present in PCR amplified (1) genomic DNA. This method requires that each oligonucleotide probe is independently labeled and that each filter containing the amplified target DNA is treated under very specific stringency conditions. These limitations become very significant when the number of genetic variations is large and when only a small number of individuals must be tested (2). The reverse dot-blot technique differs from the previous one in that the probe is the membrane anchored ligand, and the reporter moiety is attached to the DNA being tested. In this approach, DNA labeled during the amplification is simultaneously hybridized to a set of specific probes which have been previously fixed to the test membrane by virtue of a long poly dT tail (3). This method is not easily standardizable because each tailing experiment generates sequences that vary in length. In addition, most of the attached DNA is dT tail rather than specific probe. The molecular typing by reverse dot-blotting (MTRB) described here uses a more consistent and efficient approach to bind the probes to the membrane. Long polymers of the specific nucleotide sequence are prepared by a method previously described (4). The polymers bind to the membranes at high efficiency. The higher concentration of the attached probe offers the possibility of using shorter hybridization times and less sensitive detection systems or conditions than the other methods. Once filters, each containing the entire set of polymers, are prepared, the time (less than one working day) and energy involved in the typing procedure is no longer directly related to the number of sequences to be tested. In addition, the permanent source of cloned polymers offers the advantage of high reproducibility over time and between different laboratories. As an example, we present the typing of the alleles at one of the most polymorphic loci of the HLA system (2). Eight polymers, made of 17 nucleotide long probes centered on codon 57 of the DQB1 gene, were prepared. Titrated amounts (40, 16, 6 ng) of the various polymers were applied to nylon filters (FLASH, Stratagene). The target DQB1 region of the DNA to be tested was amplified with two non-radioactively (biotin) labeled primers using standard conditions (2). The PCR product was heat denatured and directly hybridized to the MTRB filters which were then stringently washed with tetramethylammonium chloride at 57°C as described (5). Both strands of the hybridized labeled DNAs were revealed with the FLASH Detection System (Stratagene) and the image recorded on an X-ray film (Figure 1). The results are captured with a flatbed scanner and a computer program automatically determines the typing, using an algorithm
based on the densities relative to the positive and negative controls present on each filter. Each of the individuals in this family was known to be heterozygous by serological means and was corespondingly positive for two of the oligonucleotide polymers. Although some cross-reaction is apparent between the allelic 0301 and 0302 sequences that differ by only one base pair, these intensities are significantly lower than the positive controls. MTRB can be easily automated and used in a reliable, costeffective screening of many different genes. In particular it will be extremely useful in testing those genes in which the known mutations are quite numerous.
REFERENCES 1. Mullis,K.B. and Faloona,F.A. (1987) Methods Enzymol. 155, 335-350. 2. Trucco,G., Fritsch,R., Giorda,R. and Trucco,M. (1989) Diabetes 38,
1617-1622. 3. Saiki,R.K., Walsh,P.S., Levenson,C.H. and Erlich,H.A. (1989) Proc. Natl.
Acad. Sci. USA 86, 8230-8234. 4. Rudert,W.A. and Trucco,M. (1990) Nucleic Acids Res. 18, 6460. 5. Wood,W.I., Gitschier,J., Lasky,L.A. and Lawn,R.M. (1985) Proc. Nati. Acad. Sci. USA 82, 1585-1588. FA1-\l.) -1;4 Is
r-A--I.
4 E ;e 6 :
:;
..
,/3.2 I
/ 4
'r.26 ,
40 16 6 ng
40 16
f>g
1 2 4
0
0
* ...
*- * ~~~~~~~~~.4
6
10
0~~~~~~~ * '0 *
SN
s & #0
&
Figure 1. MTRB method. The serological types 1.1, 1.2, 3.1 and 3.2, as shown in the pedigree of Family 5514, have been previously determined (2) and correspond to the DQB1 alleles 0501, 0602, 0301 and 0302, respectively. Rows I to 8 contain the polymers of the oligonucleotides encompassing codon 57. Row 9 contains equivalent amounts of the linearized plasmid used for polymer cloning (Bluescript II SK-, Stratagene) as a negative control. Row 10 contains an empirically determined amount of the sequence CTTCGACAGCGACGTGG, centered on codon 42, that is shared by all DQB1 alleles, as a positive control.
Rapid detection of sequence variations using polymers of specific oligonucleotides William A.Rudert and Massimo Trucco Division of Immunogenetics, Department of Pediatrics, University of Pittsburgh School of Medicine, Rangos Research Center, Children's Hospital of Pittsburgh, Pittsburgh, PA 15213, USA Submitted January 23, 1992 Hybridization with sequence-specific oligonucleotides has become the preferred method for detecting known variations present in PCR amplified (1) genomic DNA. This method requires that each oligonucleotide probe is independently labeled and that each filter containing the amplified target DNA is treated under very specific stringency conditions. These limitations become very significant when the number of genetic variations is large and when only a small number of individuals must be tested (2). The reverse dot-blot technique differs from the previous one in that the probe is the membrane anchored ligand, and the reporter moiety is attached to the DNA being tested. In this approach, DNA labeled during the amplification is simultaneously hybridized to a set of specific probes which have been previously fixed to the test membrane by virtue of a long poly dT tail (3). This method is not easily standardizable because each tailing experiment generates sequences that vary in length. In addition, most of the attached DNA is dT tail rather than specific probe. The molecular typing by reverse dot-blotting (MTRB) described here uses a more consistent and efficient approach to bind the probes to the membrane. Long polymers of the specific nucleotide sequence are prepared by a method previously described (4). The polymers bind to the membranes at high efficiency. The higher concentration of the attached probe offers the possibility of using shorter hybridization times and less sensitive detection systems or conditions than the other methods. Once filters, each containing the entire set of polymers, are prepared, the time (less than one working day) and energy involved in the typing procedure is no longer directly related to the number of sequences to be tested. In addition, the permanent source of cloned polymers offers the advantage of high reproducibility over time and between different laboratories. As an example, we present the typing of the alleles at one of the most polymorphic loci of the HLA system (2). Eight polymers, made of 17 nucleotide long probes centered on codon 57 of the DQB1 gene, were prepared. Titrated amounts (40, 16, 6 ng) of the various polymers were applied to nylon filters (FLASH, Stratagene). The target DQB1 region of the DNA to be tested was amplified with two non-radioactively (biotin) labeled primers using standard conditions (2). The PCR product was heat denatured and directly hybridized to the MTRB filters which were then stringently washed with tetramethylammonium chloride at 57°C as described (5). Both strands of the hybridized labeled DNAs were revealed with the FLASH Detection System (Stratagene) and the image recorded on an X-ray film (Figure 1). The results are captured with a flatbed scanner and a computer program automatically determines the typing, using an algorithm
based on the densities relative to the positive and negative controls present on each filter. Each of the individuals in this family was known to be heterozygous by serological means and was corespondingly positive for two of the oligonucleotide polymers. Although some cross-reaction is apparent between the allelic 0301 and 0302 sequences that differ by only one base pair, these intensities are significantly lower than the positive controls. MTRB can be easily automated and used in a reliable, costeffective screening of many different genes. In particular it will be extremely useful in testing those genes in which the known mutations are quite numerous.
REFERENCES 1. Mullis,K.B. and Faloona,F.A. (1987) Methods Enzymol. 155, 335-350. 2. Trucco,G., Fritsch,R., Giorda,R. and Trucco,M. (1989) Diabetes 38,
1617-1622. 3. Saiki,R.K., Walsh,P.S., Levenson,C.H. and Erlich,H.A. (1989) Proc. Natl.
Acad. Sci. USA 86, 8230-8234. 4. Rudert,W.A. and Trucco,M. (1990) Nucleic Acids Res. 18, 6460. 5. Wood,W.I., Gitschier,J., Lasky,L.A. and Lawn,R.M. (1985) Proc. Nati. Acad. Sci. USA 82, 1585-1588. FA1-\l.) -1;4 Is
r-A--I.
4 E ;e 6 :
:;
..
,/3.2 I
/ 4
'r.26 ,
40 16 6 ng
40 16
f>g
1 2 4
0
0
* ...
*- * ~~~~~~~~~.4
6
10
0~~~~~~~ * '0 *
SN
s & #0
&
Figure 1. MTRB method. The serological types 1.1, 1.2, 3.1 and 3.2, as shown in the pedigree of Family 5514, have been previously determined (2) and correspond to the DQB1 alleles 0501, 0602, 0301 and 0302, respectively. Rows I to 8 contain the polymers of the oligonucleotides encompassing codon 57. Row 9 contains equivalent amounts of the linearized plasmid used for polymer cloning (Bluescript II SK-, Stratagene) as a negative control. Row 10 contains an empirically determined amount of the sequence CTTCGACAGCGACGTGG, centered on codon 42, that is shared by all DQB1 alleles, as a positive control.
