Proc. Natl. Acad. Sci. USA
`Vol. 86, pp. 2766-2770, April1989
`Genetics
`
`Detection of polymorphisms of human DNA by gel electrophoresis
`as single-strand conformation polymorphisms
`(mobiUty shift of separated strands/point mutation/restrictioa fragment length polymorpbism)
`
`MASATO 0RJTA, HIROYUKI IWAHANA*, HIROSHI KANAZAWAt, KENSHI HAYASHI, AND TAKAO SEKIYA;
`
`Oncogene Division, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104, Japan
`
`Communicated by Takashi Sugimura, Dec(!mber 29, 1988
`
`ABSTRACT We developed mobility shift analysis of sin(cid:173)
`gle-stranded DNAs on neutral polyacrylamide gel electropho(cid:173)
`resis to detect DNA polymorphisms. This method follows
`digestion of genomic DNA with restriction endonucleases,
`denaturation in alkaline solution, and electrophOI"eSis on a
`neutral polyacrylamide gel. After transfer to a nylon mem(cid:173)
`brane, the mobility shift due to a nucleotide substitution of a
`single-stranded DNA fragment could be detected by hybrid(cid:173)
`ization with a nick-translated DNA fragment or more clearly
`with RNA copies synthesized on each strand of the DNA
`fragment as probes. As the mobility shift caused by nucleotide
`substitutions might be due to a conformational change of
`single-stranded DNAs, we designate the features of single(cid:173)
`stranded DNAs as single-strand conformation polymorphisms
`(SSCPs). Like restriction fragment length polymorphlsms
`(RFLPs), SSCPs were found to be alleUc variants of true
`Mendelian traits, and therefore they should be useful genetic
`markers. Moreover, SSCP analysis has the advantage over
`RFLP analysis that it can detect DNA polymorphisms and point
`mutations at a variety of positions in DNA fragments. Since
`DNA polymorphisms have been estimated to occur every few
`hundred nucleotides in the human genome, SSCPs may provide
`many genetic markers.
`
`The nucleotide sequences of DNAs in humans are not
`identical in different individuals. Nucleotide substitutions
`have been estimated to occur every few hundred base pairs
`in the human genome (1). Nucleotide sequence polymor(cid:173)
`phism has been detected as restriction fragment length
`polymorphism (RFLP). RFLP analysis of family members
`has been used to construct a genetic linkage map of the
`human genome (2, 3), and this analysis has also revealed the
`chromosomal locations of genetic elements involved in he(cid:173)
`reditary diseases such as Huntington disease (4), adult
`polycystic kidney disease (5), cystic fibrosis (6-8), Alzhei(cid:173)
`mer disease (9, 10), and Duchenne muscular dystrophy (11,
`12). Thus prenatal diagnosis of diseases such as cystic fibrosis
`is possible with RFLP probes. Recently, RFLP analysis has
`indicated specific loss of heterozygosity at particular loci on
`chromosomes in cancerous portions of tissues in several
`human cancers, including retinoblastoma, Wilms tumor,
`small cell carcinoma of the lung, renal cell carcinoma,
`bladder carcinoma, breast carcinoma, meningioma, acoustic
`neuroma (see ref. 13 for a review), colorectal carcinoma (14,
`15), and multiple endocrine neoplasia type 1- or type 2-
`associated carcinomas (16, 17). This loss of heterozygosity
`suggests the involvement of recessive mutation of particular
`genes in development of these cancers.
`Although RFLPs are very useful for distinguishing two
`alleles at chromosomal loci, they can be detected only when
`DNA polymorphisms are present in the recognition se-
`
`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked "advertisement"
`in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`
`quences for the corresponding restriction endonucleases or
`when deletion or insertion of a short sequence is present in
`the region detected by a particular probe. To identify DNA
`polymorphisms more efficiently, Noll and Collins used a
`simplified method of denaturing gradient gel electrophoresis
`(18) that had been developed by Myers et al. (19). As analysis
`of mobility shift [probably due to a conformational change of
`single-stranded DNAs on polyacrylamide gel electrophoresis
`(20)] has been used to detect point mutations (21), in this work
`we examined whether the mobility shift of single-stranded
`DNA caused by a single nucleotide substitution could be used
`to detect nucleotide sequence polymorphisms. The results
`indicated that mobility shift analysis is an efficient method for
`detecting DNA polymorphisms and for distinguishing the two
`alleles at chromosomal loci.
`
`MATERIALS AND METHODS
`Cell Lines. The human bladder carcinoma cell line T24 was
`obtained from the American Type Culture Collection. The
`human malignant melanoma cell line SK2 was established
`from a tissue that had been maintained in nude mice (22).
`DNA Isolation. High molecular weight DNA was prepared
`from human leukocytes or cultured human tumor cell lines by
`the method of Blin and Stafford (23).
`Plasmids. Plasmid pNC0106 was prepared by inserting a
`2.9-kilobase pair (kb) Sac I fragment of the HRASJ gene from
`SK2 cells into pUC19 (24). Plasmid pT22 was constructed by
`inserting a 6.6-kb BamHI fragment of the HRASJ gene from
`T24 cells into pBR322 (a gift from M. Wiglar, Cold Spring
`Harbor Laboratory).
`Subcloning and Sequencing of DNA Fragments. From
`pNC0106 and pT22, a 371-base-pair (bp) Pst I fragment
`carrying exon 1 and a 298-bp Pst I fragment containing exon
`2 of the HRASJ gene were isolated and subcloned into the
`pGEM-2 vector (Promega Biotec). The nucleotide sequences
`of the subcloned fragments were determined by the dideoxy(cid:173)
`nucleotide method (25), using Sequenase (United States
`Biochemical) and the SP6 or T7 promoter primer (Promega
`Biotec).
`Analysis of Single-Strand Conformation Polymorphisms
`(SSCPs). High molecular weight DNA (20 p.g) was digested
`completely with restriction endonucleases under the condi(cid:173)
`tions recommended by the suppliers. The reaction mixture
`was extracted once with phenol/chloroform (1:1, voljvol)
`and once with chloroform. After addition of 0.1 vol of 3M
`sodium acetate, DNA fragments were precipitated from the
`
`Abbreviations: RFLP, restriction fragment length polymorphism;
`SSCP, single-strand conformation polymorphism.
`*Present address: Department of Internal Medicine, School of
`Medicine, The University of Tokushima, 3-18-15 Kuramoto-cho,
`Tokushima 770, Japan.
`tPresent address: Department of Applied Biology, Faculty of Tech(cid:173)
`nology, Okayama University, 3-1-1 Tsushimanaka, Okayama 700,
`Japan.
`*To whom reprint requests should be addressed.
`
`2766
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`

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`Genetics: Orita et al.
`
`Proc. Nat/. Acad. Sci. USA 86 (1989)
`
`2767
`
`aqueous phase by addition of2.5 vol of ethanol. Strands were
`separated out by the method of ~~xam and Gil~ert (20) .with
`a slight modification. DNA precipitates were dtssolved m 20
`ILl of denaturing solution (0.3 M NaOH/1 mM EDTA) and
`then mixed with 3 ILl of 50% (vol/vol) glycerol/0.25% xylene
`cyanol/0.25% bromophenol blue. The mixture was applied to
`a neutral 5% polyacrylamide gel (20 x 40 x 0.2 em) with or
`without 10% glycerol in a well of 10 mm width and subjected
`to electrophoresis in 90 mM Tris-borate, pH 8.3 I 4 mM EDT A
`at 180 V for 12-36 hr at l7°C. DNA fragments in the gel were
`then transferred to a nylon membrane (Hybond-N, Amer(cid:173)
`sham) by electrophoretic blotting in 0.025 M sodium phos(cid:173)
`phate, pH 6.5, at 1 A for 2 hr at 4°C by the procedure
`recommended by the membrane supplier. The membrane
`was then dried and baked at 80°C for 2 hr. Hybridization with
`32P-labeled DNA probes was performed in 50% (voljvol)
`formamide/6X SSC (lx SSC is 0.15 M sodium chloride/
`0.015 M sodium citrate, pH 7.0)/10 mM EDTA/5x Den(cid:173)
`hardt' s solution (1 x Denhardt' s solution is 0.02% bovine
`serum albumin/O.o2% Ficoll/0.02% polyvinylpyrrolidone)/
`0.5% NaDodS04 containing denatured salmon sperm DNA at
`100 ILg/ml and 10% dextran sulfate at 42°C for 16 hr. The ~lots
`were washed twice in 2x SSC/0.1% NaDodS04 for 30 mm at
`65°C and then once in O.lX sse at 65°C for 10 min.
`Autoradiography was carried out at -8ooc for 2-7 days ~Y
`exposing the membranes to x-ray film (XAR-5, Kodak) With
`an intensifying screen (Cronex Lightning Plus, DuPont).
`Analysis of RFLP. RFLP analysis was performed. as de(cid:173)
`scribed (26). High molecular weight DNA (5~J.g) was digested
`with an appropriate restriction endonuclease and the digest
`was fractionated by electrophoresis in a 0.7% agarose gel.
`DNA Probes for Hybridization. Cloned Pst I fragments 371
`and 298 bp long carrying exon 1 and 2 of the normal human
`HRASJ gene (27), respectively, were used as specific probes
`for the corresponding exons. The 2.8-kb Hindiii fragment
`isolated from phage 9Dll (28), provided by the Japanese
`Cancer Research Resources Bank, was used as a specific
`probe for the D13S2 locus on human chromosome 13 (29).
`Probes were labeled to a specific activity of 2-10 x lOS
`cpm/ ILg by nick-translation (30) with [a-32P]dCTP (3000
`Ci/mmol; 1 Ci = 37 GBq) as a radioactive substrate.
`RNA Probes for Hybridization. Single-stranded RNA
`probes were prepared by the method ofMeltonet al. (31) with
`plasmid constructs carrying the fragments used as J?NA
`probes in the pGEM-2 vector as templates. RNA synthesis on
`each strand of the templates was carried out with T7 RNA
`polymerase (TOYOBO, Tokyo) or SP6 RNA pol~mer~se
`(Amersham) in the presence of [a-32P]UTP as a radtoactive
`substrate. Concentration of UTP was adjusted to 500 ILM by
`adding the nonradioactive nucleotide (final specific activity,
`40 Ci/mmol) to ensure synthesis of full-length RNA copies.
`The hybridization conditions and washing procedures were
`the same as those for DNA probes.
`
`RESULTS
`Mobility Shift by Single Base Substitution. To determine
`whether a single base substitution altered the mobility of
`single-stranded DNAs on neutral polyacrylamide gel electro(cid:173)
`phoresis, we separated Pst I fragments carrying exon 1 or 2
`of the human HRASJ gene, whose nucleotide sequences are
`known. In the human melanoma cell line SK2, one of the two
`alleles of the HRASI gene is known to be activated by point
`mutation at codon 61 in exon 2 (32) and also amplified about
`10-fold (33). The human bladder carcinoma cell line T24 has
`been reported to contain only one allele of the HRASJ gene,
`which carries a mutated codon 12 in exon 1 (34, 35). From
`plasmid constructs pNC0106 and pT22, containing the trans(cid:173)
`forming allele of the HRASJ gene of SK2 and T24 cells,
`respectively, a 371-bp Pst I fragment carrying exon 1 of the
`
`gene was isolated and subcloned in the pGEM-2 vector.
`Similarly, a 298-bp Pst I fragment carrying exon 2 of the
`HRASJ gene was isolated from the same plasmid constru~ts
`and subcloned. By determination of the total nucleotide
`sequences of the subcloned fragments, we confirmed the
`single nucleotide substitution at codon 12 in the 371 nucleo(cid:173)
`tides ofthe Pst I fragment between the SK2 gene and the T24
`gene (GGC in the SK2 gene and GTC in the T24 gene). ~he
`nucleotide sequences of the 298-bp Pst I fragments carrymg
`ex on 2 of the SK2 and T24 genes were also confirmed to differ
`from each other by only one nucleotide iri codon 61 (CTG in
`the SK2 gene and CAG in the T24 gene). After denaturation
`in alkaline solution, these cloned Pst I fragments were
`subjected to electrophoresis in neutral 5% polyacrylamide
`gel. The separated strands were then transferred to a nylon
`membrane by electrophoretic blotting and hybridized with
`32P-labeled DNA probes. As shown in Fig. lA, the pair of
`separated strands of the Pst I fragment carrying exon 1 of the
`T24 gene (lane 2) moved slightly faster than those of.the SK2
`gene (lane 1). In the case of the Pst I fragment carrymg exon
`2, the mobilities of the separated strands of the SK2 gene
`(Fig. lA, lane 3) were significantly different from those of the
`T24 gene (lane 4). Three bands were observed in the sample
`from the SK2 gene. Hybridization with single-stranded RNA
`probes showed that the bands with the fastest and the slow~st
`mobilities were from the same strand of the fragment, while
`the middle band corresponded to the complementary strand
`(data not shown). Usually the slowest-moving band was the
`major one from the particular strand and the ratio of the
`slowest and the fastest bands varied depending on the
`conditions of electrophoresis, especially the temperature of
`the running gels. These results suggested that a particular
`single-stranded DNA could take at least two different mo(cid:173)
`lecular shapes, depending on the conditions of electropho(cid:173)
`resis.
`In the system containing homogeneous cloned DNA frag(cid:173)
`ments, we could demonstrate mobility shift of single(cid:173)
`stranded DNAs due to a single base substitution. To deter(cid:173)
`mine whether the same mobility shift could be observed in the
`presence of DNA fragments other than a target frag!llent, we
`digested genomic DNAs from the two tumor cell hnes SK2
`and T24 with Pst I and subjected the total digests to
`electrophoresis in neutral polyacrylamide gel after denatur(cid:173)
`ation. As shown in Fig. lB, the patterns of the separated
`strands of the fragments carrying exon 1 or 2 of the HRASJ
`gene from the genomic DNAs were essentially the same as
`those of the cloned fragments. This result indicated that the
`mobility shift due to a single base substitution of a single(cid:173)
`stranded DNA fragment in total digests of genomic DNA
`
`A
`
`B
`
`2
`
`3
`
`4
`
`2
`
`3
`
`4
`
`FtG. 1. Mobility shift of single-stranded DNA fragments due to
`a single base substitution. (A) Plasmid clones (2 pg) of fragments
`carrying exon 1 (371 bp) and exon 2 (298 bp) of the HRASJ gene from
`malignant melanoma SK2 cells (lanes 1 and 3, respectively) and from
`bladder carcinoma T24 cells (lanes 2 and 4, respectively) were
`digested with Pst I. (B) Total genomic DNAs (20 ~J.g) from SK2 cells
`(lanes 1 and 3) and from T24 cells (lanes 2 and 4) were digested with
`Pst I. After denaturation, the fragments produced were subjected to
`electrophoresis in neutral polyacrylamide gel without glycerol.
`Single-stranded DNAs were transferred to a nylon membrane and
`hybridized with the 32P-Iabeled DNA probe for exon 1 of the HRASJ
`gene (lanes 1 and 2 in A and B) and the probe for ex on 2 of the gene
`(lanes 3 and 4 in A and B).
`
`GeneDX 1009, pg. 2
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`

`

`2768
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`Genetics: Orita et al.
`
`Proc. Nat/. Acad. Sci. USA 86 ( 1989)
`
`alleles, the SSCP analysis ofthe other DNA sample shown in
`Fig. 3A revealed the presence of an allele with "very slow"
`(vs) mobility in ttie fragment. The SSCPs of the other
`fragments, F3, F4, and F5, could also distinguish at least two
`alleles with "slow" (s) or "fast" (f) mobility. Analysis of 19
`DNA samples revealed that mobility shifts found in F4 and FS
`were coincidental.
`Mendelian Inheritance of SSCPs. To confrrm that the
`observed SSCPs of the Hae III fragments of the region at the
`DJ3S2 locus were due to allelic variants of true Mendelian
`traits, we analyzed the DNAs of nine individuals in two
`related families. In Fig. 3A, SSCPs of fragments F2, F3, and
`F4 and the alleles identified are indicated. In each family, the
`genotypes of the progenies were consistent with the parental
`genotypes.
`Relationship Between SSCPs and Rn..Ps. The same 19 DNA
`samples analyzed for SSCPs were also subjected to RFLP
`analysis. The DNAs were digested with Msp I or Taq I and
`RFLPs were detected by hybridization with the 32P-labeled
`DNA probe for the DJ3S2 locus. Of the 19 DNA samples
`digested with Msp I, five samples (sample 2 in Fig. 2, data not
`shown, samples 2, 3, 5, and 8 in Fig. 3B) showed RFLP. By
`Taq I digestion, RFLP was observed in only one of the DNA
`samples (sample 2 in Fig. 2, data not shown). Therefore,
`RFLP analysis revealed heterozygosity at the DJ3S21ocus in
`only 5 of 19 individuals, while with SSCP analysis heterozy(cid:173)
`gosity at the locus was found in at least one of the four Hae
`III fragments in 18 of the 19 DNA samples. This fact
`demonstrates that SSCP analysis is a superior tool for
`detection of genetic polymorphisms.
`Factors Affecting SSCP Analysis. The mobility shift of
`single-stranded DNAs with DNA polymorphisms observed
`on neutral polyacrylamide gel electrophoresis is most likely
`due to conformational variations of the molecules. The
`conformation of single-stranded nucleic acid is expected to be
`affected by environmental factors such as the temperature of
`the gel during electrophoresis, the concentration of electro(cid:173)
`phoresis buffer, and the presence of denaturing agents in gels.
`The mobility shift of the Pst I fragments carrying exon 1 of
`the HRASJ gene shown in Fig. 1A (lanes 1 and 2) was clearly
`observed on electrophoresis at 17°C but not prominently at
`23°C (data not shown). The pattern of the separated strands
`
`could be detected and was not influenced by the presence of
`a large amount of unrelated DNA fragments.
`SSCP Analysis of Human DNA at the D13S2 Locus. The
`above results encouraged us to apply the mobility shift of
`single-stranded DNA due to a single base substitution to
`detection of nucleotide sequence polymorphisms of a partic(cid:173)
`ular fragment and, as can be done with RFLPs, to distin(cid:173)
`guishing two alleles at chromosomal loci. As the mobility
`shift might be due to a conformational change of the single(cid:173)
`stranded DNAs, we designated the polymorphisms detected
`by the method as SSCPs.
`Leukocyte DNA samples from 19 individuals (10 unrelated
`and 9 in two families) were digested with Hae III, and SSCPs
`of the fragments obtained from a region of about 3 kb at the
`DJ3S2 locus on chromosome 13 were analyzed. When the
`digests were subjected to electrophoresis without denatur(cid:173)
`ation and hybridized with the 32f.Jabeled 2.8-kb Hindiii
`fragment as a specific probe for the D JJS21ocus, five distinct
`double-stranded DNA fragments (F1 to F5 in order of size)
`without any RFLP were observed in all DNA samples. The
`results on DNA samples 1 and 2 are shown in Fig. 2A as
`examples. In contrast with the double-stranded fragments,
`separated strands of the same DNA fragments showed SSCPs
`with considerable frequency. Representative results are
`shown in Fig. 2B-D. When nick-translated DNA was used as
`a probe, SSCPs were apparently observed in at least one of
`the four fragments (F2 to F5) in all four DNA samples (Fig.
`2B). The mobility shift of one of the strands of fragment F4
`in sample 1 was especially marked. However, the mobility
`shifts of single strands in other fragments were small and
`therefore the difference of the shifts was not clear when both
`strands of the fragments were hybridized with the nick(cid:173)
`translated probe. To overcome this disadvantage, RNA
`copies (RNA 1 and 2 in Fig. 2 C and D) of each strand of the
`DJ3S2 DNA fragment were prepared separately and used as
`probes for hybridization. As shown in Fig. 2 C and D, with
`either the RNA 1 or RNA 2 probe SSCPs were clearly
`detected in all fragments except fragment Fl. In Fig. 2E, the
`alleles distinguished by SSCPs are summarized. SSCPs found
`in fragment F2 by using the RNA 1 probe could distinguish
`alleles with three different mobilities, designated as "slow"
`(s), "fast" (f), and "very fast" (vf). In addition to these three
`c
`E
`D
`B
`1 2 3 4 1 2 3 4 1 2 3 4 Fra~
`Probe
`F1 - - - - -:---:--· - -
`
`A
`
`bp
`
`1 2
`
`1200--
`
`-
`
`....
`
`F2/RNA1
`
`2
`
`3
`
`s/f s/vf s/f
`
`4
`
`-
`
`f/vf
`
`650-
`
`440-
`430-
`
`310-
`
`F2 • • • • • • • - - - - -
`
`F3/RNA1
`
`F3 _,... ___ ,_...._
`~"
`F4 ____ _...._ __
`
`. ·.
`
`F5
`
`Probe: dsDNA
`
`RNA1
`
`RNA2
`
`F4/RNA2
`
`F5/RNA1
`
`f/f s/f s/s s/f
`
`s/f s/s s/s s/s
`
`s/f s/s s/s s/s
`
`FIG. 2. SSCP analysis of human DNAs at the DJ3S2locus. DNA samples 1-4 were prepared from leukocytes offour unrelated individuals
`and digested with Hae III. The resultant fragments were subjected to electrophoresis in neutral polyacrylamide gel containing 10% glycerol
`before (A) and after (B, C, and D) denaturation. DNAs in the gel were transferred to a nylon membrane and then hybridized with the 32P-labeled
`double-stranded DNA (dsDNA) probe for the DJ3S2locus (A and B) and with the 32P-labeled single-stranded RNA probes for the DJ3S2locus
`(RNA1 inC and RNA2 in D). The five fragments produced from the Dl3S2 region by Hae III digestion were designated as F1 to F5 in order
`of size. Alleles identifed by SSCPs are indicated in E with higher magnifications of informative fragments observed in C or D.
`
`GeneDX 1009, pg. 3
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`

`

`Genetics: Orita et al.
`
`Proc. Natl. Acad. Sci. USA 86 (1989)
`
`2769
`
`A
`
`Fragm~
`~Probe
`
`F21RNA1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`•
`
`sl vf
`
`fl vf vf/vf sl f vsl vf
`
`fl f
`
`fl vf vsl f
`
`f/vf
`
`sl f
`
`sl f
`
`fl f
`
`si s sl f
`
`sl f
`
`sl f
`
`sl f
`
`slf
`
`sl f
`
`sis sis sl f
`
`sis
`
`sis
`
`sis
`
`sis sis
`
`F31RNA1
`
`F41RNA2
`
`B
`
`kb 1 2 3 4 5 6 7 8 9
`
`15----------
`10.5- ww
`
`..
`
`w
`
`FIG. 3. SSCP and RFLP analyses of family members. (A)
`Leukocyte DNAs (20 J.Lg) from the family inembers indicated at the
`top (o, females; o, males) were subjected to SSCP analysis using the
`Dl3S2 probe as described in the legend for Fig. 2. As the mobility
`shifts found in fragments F4 and F5 were the same, the results with
`fragment F5 are not shown. (B) The leukocyte DNAs (5 J.Lg) digested
`with Msp I were subjected to RFLP analysis using 32P-labeled
`dsDNA as a probe for the DJ3S2 locus.
`
`of the fragments carrying exon 2 of the gene observed at l7°C
`and shown in Fig. 1A (lanes 3 and 4) was also altered at 23°C.
`Thus, the higher temperature might destroy some semistable
`conformations. The concentration of the running buffer also
`affected the mobility shift. When electrophoresis of the Pst I
`fragments analyzed in Fig. 1A was performed in a buffer of
`lower concentration (45 mM Tris-borate, pH 8.3/2 mM
`EDTA) at l7°C, the mobility shifts observed were similar to
`those at the higher temperature (23°C). Presence of 10%
`glycerol in gels also affected the mobility shift. However the
`effect of glycerol was rather complicated and mobility shifts
`due to DNA polymorphisms were often enhanced by this
`reagent. For example, the mobility shifts observed in Fig. 2
`were enhanced when electrophoresis was performed in gel
`containing 10% glycerol. On the other hand, the mobility shift
`shown in Fig. 1 was reduced by the presence of 10% glycerol
`in the gel.
`
`DISCUSSION
`By neutral polyacrylamide gel electrophoresis, we could
`separate two single-stranded DNA fragments in which the
`nucleotide sequences differed at only one position. The
`mobility shift due to a single base substitution could be
`observed not only in cloned fragments but also in fragments
`of total genomic DNA after restriction endonuclease diges(cid:173)
`tion. We applied the method to detect nucleotide sequence
`polymorphisms in human genomic DNA and could observe
`the mobility shift of single-stranded DNA by using a genomic
`sequence probe arbitrarily chosen. Single-stranded DNAs of
`
`the same nucleotide length can be separated by polyacryl(cid:173)
`amide gel electrophoresis, probably due to a difference in
`their predominant semistable conformations (20). The mo(cid:173)
`bility shift of single-stranded DNAs with DNA polymor(cid:173)
`phisms observed on gel electrophoresis might also be due to
`conformational change, and so we designated the features of
`DNAs as SSCPs. We do not know whether nucleotide
`substitution at any position in a fragment can be detected by
`SSCP analysis, but DNA polymorphisms at a variety of
`positions in a fragment could cause a difference in its
`conformation and result in change in mobility of the single
`strands on gel electrophoresis. Therefore, we thought that
`DNA polymorphism could be detected more frequently by
`SSCP analysis than by RFLP analysis, and our experimental
`results revealed that this was in fact the case. Like RFLP
`analysis, SSCP analysis is simple and does not require
`complicated instruments or specialized techniques.
`As we confirmed that the observed SSCPs were due to
`allelic variation of true Mendelian traits, SSCP analysis of
`DNA fragments could be a useful and simple method for
`elucidating the human genetic linkage map by studies on
`families. Because DNA polymorphisms have been estimated
`to occur once every few hundred nucleotides of the human
`genome (1) and SSCP analysis can reveal nucleotide substi(cid:173)
`tutions at various positions in a fragment, any restriction
`endonuclease fragment with a nucleotide length suitable for
`strand separation may provide information for distinguishing
`two alleles. Therefore, in theory, on a nylon membrane
`carrying separated strands of all possible fragments of ge(cid:173)
`nomic DNA, DNA polymorphisms at any chromosomal
`locus can be detected by repeated hybridization of the
`membrane with a variety of probes.
`SSCP analysis can also be used to locate genetic elements
`involved in hereditary diseases and to detect DNA aberra(cid:173)
`tions in human cancers. Comparison of DNA fragments from
`cancerous portions of tissues with those from normal por(cid:173)
`tions by SSCP analysis can reveal amplified alleles of
`particular genes and loss of heterozygosity at particular
`chromosomal loci. A reirtarkable advantage of SSCP analysis
`is that it can be used to detect point mutations at various
`positions in a fragment. Recently, by means of the DNA
`polymerase chain reaction (PCR), a DNA segment of a single
`cell or a single sperm has been amplified to an amount
`sufficient for analysis by hybridization (36). Our preliminary
`result suggested that SSCP analysis of DNA segments am(cid:173)
`plified by PCR technique could be useful for diagnosis of
`genetic aberrations.
`
`This work was supported in part by a grant-in-aid from the Ministry
`of Health and Welfare for a Comprehensive 10-Year Strategy for
`Cancer Control, Japan, and a grant from the Special Coordination
`Fund of the Science and Technology Agency of Japan. M.O. and H.l.
`were recipients of Research Resident Fellowships from the Foun(cid:173)
`dation for Promotion of Cancer Research.
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