List of Publications

Gupta V, Arora R, Ranjan A, Bairwa NK, Malhotra DK, Udhayasuriyan PT, Saha A, Bamezai

R. Gel-based nonradioactive single-strand conformational polymorphism and mutation

detection: limitations and solutions. Methods Mol Bioi 2005;29:247-261.

Gupta V, Arora R, Saba A, Dhir A, Kar P, Bamezai R. Novel variations in the signal peptide

region of transforming growth factor PI gene in patients with hepatitis: a brief report from India.

Internat J Immunogenet (In press).

Saba A, Bairwa NK, Ranjan A, Gupta V, Bamezai R. Two novel somatic mutations in the

human interleukin 6 promoter region in a patient with sporadic breast cancer. Eur J

Immunogenet 2003;30:397-400.

Saba A, Gupta V, Bairwa NK, Malhotra D, Bamezai R. Transforming growth factor-betal

genotype in sporadic breast cancer patients from India: status of enhancer, promoter, 5'­

untranslated-region and exon-lpolymorphisms. Eur J Immunogenet 2004;31:37-42.

Saba A, Dhir A, Ranjan A, Gupta V, Bairwa N, Bamezai R. Functional IFNG polymorph isms

in intron 1 in association with an increased risk to promote sporadic breast cancer.

Immunogenetics (In press).

Awards received

Received 'Young Scientist' award for the year 2004 in the field of 'Medical Genetics' by Indian

Society of Human Genetics for the paper' Status of some of the genes involved in viral

clearance and host pathogenesis in chronic hepatitis B infection' .

Presentations made

Platform presentation made for the paper title 'Status of some of the genes involved in viral

clearance and host pathogenesis in chronic hepatitis B infection' at 29th Annual Conference of

Indian Society of Human Genetics (ISHG 2004) held in Bangalore.

Same paper was also presented at Biosparks 2004, 2nd Annual Research Festival, School of Life

Sciences, JNU.

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TI""'I aaua; No. of Pages: 4 Copy-editor: kUy Angel

i Novel variations in the signal peptide region of transforming growth factor ~~ ~1 gene in patients with hepatitis: a brief report from India ~ ~i

D

V. Gupta,* R. Arora,* A. Saha,* A. Dhir,* P. Kart & R. Bamezai*

Summary Mutations in the signal peptide region, in general, are known to inhibit cleavage by signal peptidase or cause a defective secretion of the protein to extracellular location. A preliminary analysis of genotypic status of the signal peptide region ofTGF-fH in 53 viral hepatitis patients and 90 controls using SSCP and/or direct sequencing showed a significant difference in CIC-genotype frequency at +29 position (codon 10) between a range of viral hepatitis patients and controls (P = 0.009, OR = 3.15, 0=1.29-7.678). It was observed that out of a heterogenous pool of hepatitis patients., this significant difference between patients and controls was contributed by those who were infected with HBV alone or HBV + lIDV infection (P = 0.003, OR = 5.0, 0= 1.78-13.97). Two novel variations, +29T > G in two and 36-37ins(CTGh in one patient were detected. These were not detected in 90 control samples. It is proposed that the observed variations in signal peptide region could lead to a change in the expression levels ofTGF~1. Because TGF~1 is an important candi­date for liver apoptosis, immunomodulation and liver regeneration following viral infection, a variation in genotype affecting expression could affect an individual's susceptibility to HBV related hepatitis.

Introduction VIrtually all prokaryotic and cukaryotic proteins that are destined for export from the cytoplasm are synthesized with N-terminal peptide extensions. These extensions, known as signal peptides, are essential for the initial mem­brane translocation step of export (Saier et a/., 1989). The signal peptide earmarks a protein for transport; its physi­cal properties dictate several interactions between the exported protein and the lipids and proteins of the export

• National Centre of Applied Human Genetics. School of Life Sciences. Jawaharlal Nehru University. and t Department of Medicine. Maulana Azad Medical College. New Delhi. India

Received 21 May 2004; revised 16 August 2004; accepted 28 October 2004

Correspondence: R. N. K. Bamezai. National Centre of Applied Human Genetics. Human Genetics Section. School of Life Sciences. Jawaharlal Nehru University. New Delhi-110067.lndia. Tel: 91-11-26704518; 26103211; Fax: 91·11·26103211; E-mail:oamezaiOhotmail.com; bame02OOOmail.jnu.8C.in; bamezaiOncahg.org

pathway. Mutations in human signal peptides have been correlated with defective secretion and a consequent pathological state in a few reported cases (Brennan et at, 1990; Daly et Ill., 1990; Arnold et al., 1990; Watzke et al., 1991; Racchi et al., 1993; Regis-Bailly et al., 1995; Hughes et al., 2000; Yamagishi et al., 2003; Watanabe et al., 2003; Olristensen et al., 2004). An analysis there­fore of the variation in the signal peptide region of a candidate gene with a possible involvement in a disease development could provide useful information about the role of the gene in the pathogenesis of the disease.

1here have been reports of association of TGF~1 in viral hepatiris.ln hepatitis C, TGf-IH C/C-genotype at +29 position (codon 10) has been shown to be associated with resistance to antiviral therapy (Vidigal et al., 2002) and variations at codon 10 and 25 associated with fibrotic progression in hepatitis C (Gewaltig et al., 29(2). TGF~ 1 is a pleiotropic cytokine with activities ranging from arrest of liver cell regeneration and induction of apoptosis in liver diseases (Lin & Chou, 1992; Oberhammer et al., 1992; Takiya el al., 1995; Cain & Freathy, 2001) to liver necroinflanunation, fibrosis (Nagy et al., 1991; Murawaki et al., 1998) and immunomodulation (Chen et al., 1999; Mouri et al., 2002) in viral hepatitis. We have chosen TGF~ 1 as a candidate gene to study its signal peptide region for polymorphisms and a possible association with a susceptibility to a range of viral hepatitis with different independent phenotypes related to liver as an organ.

Materials and methods

Study subjects

A total of 90 (31 females and 59 males in the age range of 21-53 years) healthy controls with no previous reports of liver-related disorders and 53 (21 females and 32 males in the age range of 23-60 years) patients with viral hepatitis (i.e. the patients who showed present or previous evidence of liver disease characterized by increased serum ALT levels and/or presence of other liver-related disorders like jaundice, cirrhosis, portal hypertension, hepatic encepha­lopathy and ascites) from India with similar ethnic back­grounds were chosen for the study. Out of these, five were positive for HAV (hepatitis A virus), 21 for HBV (hepatitis A virus), three for HCV (hepatitis C virus), five for coin­fection with HBV and HDV (hepatitis delta virus) and 19

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2 V. Gupta et al.

for HEV (hepatitis E virus). These cases were confirmed to belong to different categories on the basis of the specific ELISA (enzyme-linked immunosorbent assay) or PCR (polymerase chain reaction)-based assays used commer­cially. Blood samples of 5-10 mL were collected from the subjects with informed consent. Genomic DNA extrac­tion was carried out with a standard protocol (Kunkel et at., 1977) used routinely in our laboratory.

Polymorphisms studied

Primers were designed to amplify partial5'lJfR and par­tial coding sequence containing full signal peptide region of TGF~l. The forward primer: 5' -ClC cac acc agc cct gtt cg-3'(20-mer) and the reverse primer: 5'-agc cgc agc ttg gac agg at-3'(20-mer) amplified a region between -55 to +176 (as per HUGO n<fmenclature from the translation initiation site) and gene~ated an amplicon of 231 bp.

Genotyping ,

A PCR-SSCP-~ .,f:" (Hongyo" ai, 1993) '"" carried out for the p . . ary mutation analysis. Silver stained gels were for the variant bands. The ss normal and variant D bands were eluted, amplified and cloned in pGEM-T cloning vector (Prornega, USA). The cycle sequencing of clones was carried out using the ABI Prism BigDye Tenninator cycle sequencing system (PE Applied Biosystems, Foster City, CA, USA). Direct sequencing of the PCR products of samples showing variant bands during SSCP analysis further confirmed the variations. Both· forward and reverse primers were used for sequencing of PCR products.

Statistical analysis

The differences in genotype frequencies of TGF-lil between patients with viral hepatitis and age and sex matched controls were assessed using FISher's test. The relative risk was assessed by calculating the odds ratio. For all statistical tests, the significance level adopted was P < 0.05. The statistical analysis was carried out by using the software, SPss Version 10 (SPSS, Chicago m, Illinois, USA). Powers of the associations were calculated for one­sided test using the software available at the Internet site http..JIwww.pasreur-kh.org/-glaziou/sampsizels3.htmI#a:p.

Results and discussion The amplified region involving the partial5'lJfR and the complete signal peptide region of TGF~1 was screened for the presence of variations in a range of viral hepatitis patients (as mentioned in Materials and methods section) and controls. The details of the genotypes in controls and patients are shown in Table 1. Controls as well as patients showed known polymorphisms (Cambien et at., 1996), T > C at position +29 (codon10, Leu> Pro) and G > Cat position +74 (codon25, Arg > Pro) (Fig. 1). Statistical analysis revealed a significant increase in C/C-genotype

Table 1. Number of individuals studied and the nature of TG~ 1 genotypeS' in the signal peptide region

Viral hepatitis Controls (90) patien1S (53) HBVand

Genotype No.(%) No.(%) HBV+HOV

+29, TIT 32 (35.6) 14(26.4) 6(23.1) +29, TIC 48(53.3) 21 (39.6) 9(34.6) +29, ctc 10(11.1) 15(28.3)' 10 (38.5)" +29, C/~ 0 2 (3.7) 0 36-3TrnslCTGl,b 0 1 (1.8) 1(3.8) +74, GIG n(856) 43(81.1) +74, G!C 13 (14.4) 10 (18.86)

• Nomenclature used according to HUGO; • Novel variations; , Comparison with controls gave Rsher's exact P-value as 0.009, OR = 3.15, CI = 1.29-7.69;" Comparison with controls gave Fisher's exact fI.vaIue as 0.003, OR = 5, CI = 1.78-13.97.

frequency in viral hepatitis patients as compared to controls (P = 0.009, Odds ratio = 3.15, CI = 1.29-7.69) (Table 1). The power of association calculated was 82.56%. The change of Leu to Pro at codon 10 in leader peptide has been shown to be associated with increased expression (Dunning et at., 2003). The observation of an association of the high producer genotype of TGF~1 is further supported by an observation of increased TGF~l serum levds in hepatitis (Murawaki et at., 1998). Because, TGF~1 is known as an immunosuppressor (Chen et at., 2001; Mouri et at., 2002), it could help in the establish­ment of viral infection in cases of viral hepatitis. This preliminary observation possibly suggests that individuals with 'high producer'-TGF~1 genotype may be more sus­ceptible to viral hepatitis. Apparently, it was intriguing to find that a diverse group of liver disease phenotypes showed a significant correlation with the CI C-genotype at Codon-l0, the signal peptide region, of TGF-IU gene. It was pertinent therefore to examine the association sepa­rately in each group of hepatitis, which showed a signific-ant difference only for patients infected with either HBV or HBV + HDV (P = 0.003, odds ratio = 5.00, a = 1.78-13.9) (Table 1) with the power of association as 90.94%. The observation, however, needs to be established on a larger sample size in future.

A novel variation T > G at position +29 (codonl0, Leu> Arg, Ace. No-AY576687) was observed in a hepa­titis C and a hepatitis E patient (Fig. 1). Further, an inser­tion of (CfGh was observed in a hepatitis B patient at position +36 leading to a change in trinucleotide repeat region from (CfG)s to (CfG), and an insertion of two Leu residues in the signal peptide region of TGF~l gene (Ace. No-AY576688). The functional implications of these novel variations found in the present study are yet to be elucidated. However, there are some indirect evidences of the role of these variations. The variation, Leu > Arg, is known to result into the substitution of a hydrophobic amino acid by a charged residue in the hydrophobic core region of the signal peptide of TGF~1. Further, decreas­ing the hydrophobicity of the hydrophobic region has

C 2005 Blackwell Pubtishing Ltd, IntematlOll8l Journal of Immunogenetics 32. 000-00O

Genotype at codon to TIC TIT TIC Ctc CIG TIC

Genotype at codon 2$ GIG GIG G/C GIG GIC GIC

(a)

* +29T * +29G * +74G GO:> G G G·' G G

* +29C . G G G G '. '. c . C G GG G G

~ 3~7(CTG)~ *+74C

G GG G GC G G G G' G GG

f"igwe 1. Variations 1*) in the signal peptide region of TGF~1 gene as observed in (a) SSCP and IbI after sequencing. shown through the partial chrom&tograms.

been snggrsred to nuIlify signal peptide capability and result in an expon defect (Briggs & Gierasch, 1986; Storms & Rutisbauscr, 1998). Similar findings in other proteins such as humanin, the Leu9-Leu12 region has been shown to playa functional role as a hydrophobic core and subsitu­tion of Leu by kg leads to defects in self-secretory activity of humanin (Yamagishi et aI., 2003). The functional role of the insertional mutation of two Leu resides in the signal peptide region observed in this study could further be supported by citing an example of an insertion I deletion polymorphism in the signal peptide of the human apoli­poprotein B gene that predicts two apolipoprotein B sig­nal peptides: one that encodes a peptide of 27 residues and the other a peptide of only 24 residues (VJSvikiset aI., 1990). This variation has been shown to confer a secretion defec­tive phenotype in apoB (Talmud et aI., 1996). In addition to causing a defective protein transport, the length varia­tions in the signal peptide may affect the mRNA stability.

Such a phenomenon has been shown in case of ermC gene where a mutation in the leader peptide region showed a change in mRNA stability, leading to a change in its tranS- [;] .-lation efficiency (Hue & Bechhofet; 1991). It is quite likely that the variations observed in this study in the signal peptide region of the TGF-fU gene could provide suscep­tibility to suffer from viral hepatitis at least in some cases, although the mechanism involved in the pathogenesis needs further elucidation.

Acknowledgements The financial assistance from University Grants Commis­sion (UGC) to the National Centre of Applied Human Genetics, New Delhi and the fellowship support provided to v.G., A.S. by the Council of Scientific and Industrial Research (CSIR) and to R.A., A.D. by University Grants Commission (UGC) are acknowledged.

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4 V. Gupta et al.

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Ikaman,S.O.,Myles, T.,Peach,R.j,Dooaldson,D.&Geocge,P.M. (1990) Albumin redhill (-1 Atg, 320 Ala - The): a glycopcoo:in variant of hwnan serum albumin whose pn:cucsoc has an aberrant signal peptidase cleavage site. ProceeJmgs of the Nationill Academy of Scienas United States of America, 87, 26.

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Oten, R..I-L, <liang. M.e., So, Y.H., Tsai, Y.T. & Kuo, M.L (1999) InIuIeukin-(; inhibits ttansfonning growth ~induced apoptosis through the p~ 3-kinase1Akt and signal transducers and activators of transcription 3 pathways. JOfn1IIIl of BiologiaJI Chemistry, 274, 23013.

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0nisImsen, J.H., Siggaard, e., Corydon, T.J., deSanctis, L., Kovacs. L., Robeiuon, G.L., Grqersen,N. & Ritti&s. (2004) Six no¥d mutations in the arginine YaSOpftSSin gene in 15 kiDdmIs with autosomal dominant familial neurohypophyscal dia~ insipidus give further insiPt into the pathognesis EMropetm JOIIrIftIl of Ebmrtm Genetics, 12, 44.

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Dunning. AM., Ellis, P D., McIIridc, s., Kincbenloh.t, H.L, Healey, c.s., Kemp, P.R. dill. (20031 A transforming growth factor- PI signal peptide variant incmascs secretion in vitro and is associated with increased incidmce of invasive breast canar. Ctmar Research, 63,2610.

Gewaltig, J., ~ K., Gartung, c., Bicsterleld, S. & Grcssner, A..M. (20021 Association of pol)'IllCllphisms of the transforming growth faaoc-lll gene with the rate of progieWon of HCV-induced IMc fibrosis. 0inicaJ ChimU:a Aaa, 316, 83.

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Hughes, A.E., Ralston, S.H., Macken,J., Bell, C., MacPherson, H., Wallace, R.G.H. et al. (2000) Mutations in lNFRSFI1A, affectiDg the signal peptide of RANK, causc familial c:xpansile osteolysis. Nimtu Genetics, 24, 45.

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Murawaki, Y., NlShimura, Y., Ikuta, Y., Idobe, Y., Kitamura, Y. & Kawasaki, H. (1998) Plasma transforming growth factor-lll concentrations in patients with chronic vical hepatitis. JOImIIIl of Gastroenterology and Hepato/ogy, 13, 680.

Nagy, P., SchaH, Z. & Lapis, K. (1991) Immunohistochemical detection of transforming growth factoc-lll in filxotic liver diseases. Hepatology, 14, 269.

Obechammec, F.A.., Pavelka, M., Sharma, S., TJefenbachec, R., Pucchio, A..F., Buesch, W. & Schulte-Hennann, R. (1992) Induction of apoptosis in cultured hepatocytes and in regressing liw:r by transfonning growth factor ~l. Proceedings of the National AaIdemy of Sciences of the United States of America, 89,5408.

Racchi, M., Waake, H.H., High, K.A.. & livdy, M.O. (1993) Human coagulation factor X deficiency caused by a mutam signal peptide that blocks cleavage by signal peptidase but not targeting and traDsIocarion to the endoplasmic reticulum. JOllmilI of Biologiazl Chemistry, 168, 5735.

Regis-BaiDy,A., Foumiec, B., Steinmetz,J, Gueguen, R., Siest, G. & Vasvikis, S. (19951 Apo B signal peptide insertionIddetion poIymcxpbism is involved in postprandiallipoparticles' responses. AIberosderosis, 118, 23.

Saic:r,M.H., Wemcc,P.K. &Mullec, M. (19891 Inscrtion of proteins into bacterial membranes: mechanism, characu:ristics and comparisons with the eucaryotic process. MiaobioIical. Rntiews, 53,333.

Storms, s.D. & Rutisbausec, U. (1998) A role for polysialic acid in nueral cell adhesion molecule heterophilic binding to ~ JOfn1IIIl of Biologiall Chemistry, 273, 27124.

Takiya, S., Tapya, T., Takahashi, K., Kawashima, H., Kamiya, M., Fultuzawa, Y., Kobayashi, S., Fukatsu, A.., Katoh, K. & Kakumu, s. (19951 Role of transforming growth factor- PI on hepatic rqencration and apoptosis in liver diseases. Jotn7fIIl of 0inicaJ Pathology, 48, 1093.

Talmud, P., Lins, L & Brasseuc, R. (1996) Predicrion of signal peptide fwlctional properties: a study of the orientation and angle of insertion of yeast invertase: mutants and human apolipoprotein B signal peptide variants. Protein Enginnering, 9, 317.

Vidigal, P.G., Gennec,J.J. & Zein, N.N. (20021 Polymorphisms in the intecleukin-l0, tumor necrosis factor-alpha, and transforming growth factOl'"IU genes in chronic hepatitis C patients treated with interferon and ribavirin. JOllmilI of Hepatology, 36, 271.

VJSVikis, S., Chan, L, Siest, G., Drouin, P. & Boerwiokle, E. (1990) An insertion deletion polymorphism in the signal peptide of the human apolipoprotein B gene. Human Genetics, 84, 373.

Watanabe, R., Ishibashi, T., Saitoh, Y., Shichishima, T., Maruyama, Y., Enomoto, Y. et al. (20031 ~ulier syndrome with a homozygous 13 base pair deletion in the signal peptide<Odiog region of the platelet glycoprotein Ib (~) gene. Blood Coagulation Fibrinolysis, 14,387.

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Yamagishi, Y., Hashimoto, Y., Naikuca, T. & Nishimoto, I. (2003) Identification of essential amino acids in Hurnanin, a ueuroprotectivc factor against Alzheimec's disea&e-rdevant insults. Pe(1tides, 24, 585.

C 2005 Blackwell Publishing Ltd. International Journal of Immunogenetics 32. 000-000

23 ______________________________ _

Gel-Based Nonradioactive Single-Strand Conformational Polymorphism and Mutation Detection

Limitations and Solutions

Vibhuti Gupta, Reetakshi Arora, Anand Ranjan, Narendra K. Bairwa, Dheeraj K. Malhotra, P. T. Udhayasuriyan, Anjana Saha, and Ramesh Bamezai

Summary Single-strand confonnation polymorphi~m (SSCP) for screening mutations/single­

nucleotide polymorphisms (SNPs) is a simple, cost-effective technique, saving an ex­pensive exercise of sequencing each and every PCR reaction product and assisting in choosing only the amplicons of interest with expected mutation. The principle of detec­tion of small changes in DNA sequences is based on the changes in single-strap.d DNA confonnations. The changes In electrophoretic mobility that SSCP detects are sequence­dependent. The limitations faced in SSCP range from the routine polyacrylamide gel eiectrophoresis (PAGE) problems to the problems of resolving mutant DNA bands. Both these problems could be solved by controlling PAGE conditions and by varying physical and environmental conditions such as pH, temperature, voltage, gel type and percentage, addition of additives or denaturants, and others. Despite much upgrading of the technol­ogy for mutation detection, SSCP continues to remain the method of choice to analyze mutations and SNPs in order to understand genomic variations, spontaneous and induced, and the genetic basis of diseases.

Key Words: SSCP (single-strand confonnation polymorphism); PCR (polymerase chain reaction); PCR-SSCP; PAGE (polyacrylamide gel electrophoresis); polymorphism; chemical mutagenesis; SNP (single-nucleotide polymorphism); ml":ltion detection.

1. Introduction

1. 1. What is SSCP?

The study of genomic variations has become important in understanding human history and disease susceptibility. A variety of techniques are described in the litera­ture that can detect single base mutations, insertions, and deletions, and all of these have one or the other advantage. In recent years, high-throughput methods like solid

From; Methods in Molecular Biology, vol. 291, Molecular Toxicology Protocol; Edited by; P. Keohavong and S. G. Grant © Humana Press Inc., Totowa, NJ

247

248 Gupta et al.

phase mini sequencing, detection of dissimilarly sized extension fragments by matrix­assisted laser description ionization-time of flight mass'spectroscopy (MALDI-MS), mismatch cleavage detection, oligoarray hybridization, molecular beacon signaling, fluorescence monitoring of polymerase chain reaction (PCR), electronic dot-blot assay, and denaturation high-performance liquid chromatography are already in use (for review, see ref. 1). Nevertheless, one of the commonest, simplest, and also rea­sonably sensitive methods for rapid detection of gene mutations is that of single-strand conformation polymorphism (SSCP). It has been useful in finding associations with a number of diseases ranging from hemophilia A (2), Parkinson's disease (3), hereditary spastic paraplegia (4) to schizophrenia (5) and cancer (6). Also, the technique has been useful in detecting induction of mutations in DNNcDNA in toxicology studies. Chemically induced mutagenesis has been studied using the SSCP technique in rats (7-10), humans (11-13), and cell lines (14).

SSCP relies on the ability of a single (or multiple) nucleotide change(s) to alter the electrophoretic mobility of a single-strand DNA molecule under native (nondenaturing) conditions. Under nondenaturing conditions, most single-stranded DNA molecules assume one of more stable 3D conformations that depend on the nucle­otide sequence. This change in a single nucleotide leads to a conformational change that is reflected in the electrophoretic mobility of the polymorphic sequence in com­parison with the more common "wild-type" sequence (Figs. 1 and 2).

Mutations/polymorphisms occur within many different regions of a gene (promoter, 5' untranslated region [5' UTR], exons, introns, exon-intronjunctions, 3' UTR, and oth­ers), and are screened by the following steps: (1) designing primers complementary to the region flanking the specified region; (2) subjecting the amplicons produced by PeR to SSCP analysis; (3) eluting the variant bands for characterization through sequencing; and (4) using these bands (DNA) for comparison in further analysis. A large number of samples can be prescreened for mutation(s) and single-nuleotide polymorphisms (SNPs).

The basic protocol of SSCP described by Orita in 1989 was based on PCR (15) or restriction fragment length polymorphism (16) -based detection of mutations in radio­labeled fragments on a sequencing apparatus. This basic protocol has undergone dras­tic changes since then, and nonradiolabeled (17) and fluorescence-based detection (18) has subsequently been used for SSCP analysis.

The advantages of the SSCP technique are that it is easy and cost-effective and requires low technical input. It can be used as a basic tool to identify variations before sequencing. If properly standardized, it can also be applied to find loss of heterozy­gosity (LOH) and micro satellite instability (MIS). Another advantage is the ease of separation, direct elution, and cloning of the variant band in both heterozygous and homozygous mutant situations. However, among its disadvantages are that it is at times difficult to standardize. SSCP results may be misleading if nonspecific bands are seen in some gels, a problem that can be overcome by repeating and obtaining reproducible results. Some mutations may ]lot be resolved at all using SSCP. Also, it is a low­throughput technique: at the most, a worker can analyze 30-40 samples in a day.

In the literature, a variety of parameters have been found to improve the efficiency of the SSCP technique. Additives (such as sucrose, glycerol [19J, urea [20,21J.

SSCP: Limitations and Solutions 249

formamide [20), and [22) polyethylene glycol [23), use of shorter length fragments for analysis (24), varying acrylamide percentage (25) and acrylamide to bis-acrylamide ratio (20), optimization of PCR conditions (26), use of acrylamide substitutes (such as the mutation detection enhancer [MOE] [27), PHAST SYSTEM [28,29), and agarose [30), varying pH, current, and voltage (31), use of multitemperature SSCP (n), use of cold SSCPs (33), and use of combined SSCP/duplex analysis by capillary electro­phoresis (34) have all been proposed to improve the resolution of single strands of the denatured double-stranded DNA

t .2. Standardizing SSCP It is important to remember that the optimum SSCP conditions for a particular frag­

ment are determined by sequence, i.e., the length, base composition, and type of muta­tion to be studied. At least 90% of single-base pair substitutions can be detected if the fragment length is within the optimal. size range of 130-320 bp. Also, 80% of single base pair substitutions can be detected if the PCR products are kept under 400 bp in length (35). Low pH greatly increases the sensitivity of the mutation detection and allows the screening of fragments of 800 bp in length (20), although in our experience, amplicon length remains a limiting factor.

Factors important for standardization are:

1. Gel temperature: temperature is an important factor as it affects the stable DNA confor­mation as well as the mobility. As the temperature increases, the mobility also increases, and the total run time decreases. Low temperature is maintained either by running the gel in a cold room or by circulating cold running buffer in the gel apparatus. Running the same sample at different temperatures maximizes the chances of detecting mutations, as the variations missed under one temperature may be picked up at another. The same se­quence can show two absolutely different profiles at two different temperatures. Tem­perature is one of the most important factors affecting the resolution of the single-strand DNA bands in the gels. The temperature range of 4°C (cold room) to 25-30°C (room temperature) shows the best differentiation of the single-stranded (ss)DNA bands. Migra­tion of the bands is directly related to the incre.ase in temperature.

2. Gel pH: generally, SSCP gels are run in IX TBE solution, pH 8.3. However, TBE con­centrations can be varied from 0.089 M to 134 M TBE. pH can also be lowered by adding 5-10% glycerol. Low pH can enhance sensitivity for longer fragments (20). pH can also be "arled by the addition of sucrose, formamide, polyethylene glycol (PEG), and so on. Running buffer pH can also be lowered to 6.8.

3. Voltai'e: high voltage increases mobility and hence lowers the run time. However, the heat produced at higher voltage can lead to uneven heating of the gel matrix, which can result in band smiling or frowning, if the heat dissipation system is not effective.

4. Gel matrix: bands move more slowly and resolve better at higher acrylamide concentra­tions. The ... gel % is mainly determined according to the size of the sequence to be ana­lyzed. Sometimes the use of alternative matrixes like MOE, gene amp, Hydrolinks, agarose, or PHAST can also increase the possibility of finding a mutation.

5. Additives: sucrose (10%), urea, glycerol (5-10%), PEG, ethylene glycol, or formamide may be added to gel. It has also been suggested that 5-10% glycerol stabilizes the 3D DNA conformations, especially at higher temperature, although in our experiments we have not found any difference in resolution of bands. Glycerol enhances the mutation

250 Gupta et al.

~.

~. • 0- •••••••••••••••••••• ~ . ........... .

Homozygous profile Heterozygous profile

1 Denaturation 1

...................... ..' .............

~ ..................................... , .......... --k ....................... .

Single strand separation after denaturation

(Continued next page)

Fig. 1. Schematic representation of hypothetical conformation differences adopted by the mutation-bea,ring ssDNA molecules (''k) in comparison with the wild-type (nonmutant) ssDNA molecule, thlls resl.\ltin~ in the mobility shifts of the two strands of DNA. The heterozygous profile in some cases may only resolve into three bands instead of four.

detection and interpretation of bands, as it reduces deformer formation and is known to affect pH ill the TBE buffer system (19).

6. Another option is to label both strands. Using different colors for each strand allows the detection of the residual double-stranded molecules remaining after denaturation and also overlapping peaks in the two colors may confirm the presence of unresolved bands in the three-band situation. Dedicating a color to each fragment allows one to confirm the origin of extra peaks.

It is difficult to predict optimum conditions for a particular fragment even if its complete sequence is known. Optimum conditions have to be standardized by trial-

SSCP: Limitations and Solutions

1

Homozygous profile

(two DNA bands)

Snap chilling and gel analysis

22

Fig. I (Continued)

1

t···) . ....... .. : ......... :... ; ..... ~ .. : .... ,: ~....- ... ~.

\.r····..: ..... _ .. ) : ..... .... :

:'{:( .............. /

251

Heterozygous profile

(four DNA bands)

~ ) (

, llll l'IiC()Il'" Thl' l'l(l1cilil(ll1' :tIL' i ~i ( ,ll I, I" !\L

l\I lel

fur

[ld

II ILL!' ,111ltatJOll\ ('[ I !lIlt jllIl III It..: elllJ

, ' he I",' ( ;;1 1()1 j ()1i

,,! IIll d!hl

II ell t lie' IL'111 IlL III

lildll l ] r'l'i'l'l'

he \1" [illd cilile' :11

6

SSCP: Limitations and Solutions 253

7. TBE buffer: 80 mM Tris-base. 40 mM boric acid. 2 mM EDT A. Make as lOX stock solu­tion and store at room temperature.

8. Ethidium bromide: 10 mglmL stock solution in autoclaved distilled water (use gloves and protect from direct light; store at 4°C).

9. 1% Agarose in IX TBE buffer.

2.2. Polymerase Chain Reactions Use gloves to handle.

I. Sterile O.5-mL PCR tubes and microtips. 2. PCR kit (reaction buffer. enzyme. dNTP mix; store at -20°C). 3. Primers for the region to be studied (store at -20°C until use and after). 4. Mineral oil (store at room temperature). 5. Thermal Cycler (e.g .• MJ Research. Miami. FL).

2.3. Polyacrylamide Gel Preparation and Gel Eletrophoresis I. Autoclaved distilled water. 2. Glass plates of 20 x 18 cm. spacers. and combs of I-mm thickness each. with a well size

of 3.5 mm. 3. Gel electrophoresis system. 4. Acrylamide: bis-acrylamide stock (29: I); store at 4°C. 5. 10% APS solution and TEMED: 6. TBE running buffer: see Subheading 2.1.. item 7. 7. SSCP dye: 0.15 g Ficoll. 0.0025 g bromophenol blue. 0.0025 g xylene cyanol. 950 ilL

formamide. and 50 ilL IX TBE. 8. Power pack (e.g .• Bio-Rad PAC 3000. Hercules. CA).

2.4. Silver Staining of SSCP Gel I. Fixative: 10% ethanol and I % acetic acid solution. 2. Staining solution: 0.1 % silver nitrate solution. 3. Developing solution: 1.5% NaOH solution. 4. 0.75% Sodium carbonate and X% acetic acid solution.

2.5. PAGE Elution of DNA I. Elution buffer preparation (for 25 mL): add 0.9365 g ammonium acetate and 0.0536 g

magnesium acetate to 10 mL water and autoclave. Add to this 125 ilL C 2 M EDT A and 250 !-lL of 10% SDS and make up the volume to 25 mL.

2.6. Agarose Purification of DNA Bands I. Agarose (molecular biology .grade; e.g .• Sigma or Pronadisa. Rehovat. Israel). 2. Tris-HCI-~aturated phenol. pH 8.0 (store at 4°C). 3. 3 M Sodium acetate. pH 5.2 (store at room temperature). 4. Absolute ethanol (e.g .• Merck. Mumbia. India).

2.7. Ligation of DNA Bilnds for Sequencing I. PCR product ligation kit (e.g .• Promega. Madison. WI): 2. Insert DNA.

254 Gupta et al.

2.8. Transformation of Ligated Product and Screening of Positive Clones 1. Competent bacterial cells (e.g., DH5a). 2. Sterile culture plates. 3. Luria broth medium (e.g., Himedia, Mumbai, India). 4. Ampicillin (store at -20°C).

3. Methods (see Note 1)

3.1. Preparation of Template 1: Incubate blood (5 mL) with 4.5 mL lysis buffer for 30 min on ice. Break the clumps or

clots with the help of a Pasteur pipet. 2. Centrifuge at 4°C for 20 min at I100g. 3. Resuspend the pellet gently in 4.5 mL of digestion buffer. 4. Add Proteinase K solution to the homogenous cell suspension at a final concentration of

100 ng/mL. 5. Incubate the suspension at 65°C for I h, followed by overnight incubation at 37°C with

gentle shaking. 6. Next day, de proteinate the cell suspension by-extracting twice with an equal volume of

phenol/choloroform and twice with chloroform, centrifuging at 2200-3500g for 20 min at room temperature.

7. Precipitate by adding 1/ 10th vol of 3 M sodium acetate and 2 vol of ethanol. 8. Spool DNA by binding on a glass rod or pick DNA in a broad-mouthed Pasteur pipet anc

wash with chil!ed 70% ethanol. 9. Dry the DNA and dissolve in TE.

10. Mix the genomic DNA with 1/6th vol of gel loading dye and load into wells of a 0.8% agarose gel. Run at low voltage of 40-45 volts. Visualize on a UV transilluminator. The high-quality genomic DNA should be seen as a band without shearing.

11. Quantitate by taking the optical density (OD) at 260 nm. Also compare the ratio of OD at 260 nm with that at 280 nm to check the quality; DNA samples having OD ratios> 1.5 are considered to have low protein contamination. Dilute the samples to the concentration of 25 ng/IlL in autoclaved distil:ed water, and store at -20°C until analysis.

3.2. Polymerase Chain Reaction for Amplification of the Region to Be Studied 1. A 12.5 !!L vol of PeR reaction for each sample includes: 6.2.5 pmol of each primer, lOng

target DNA, 100 nM dNTPs, IX peR reaction buffer, 0.5 U To.q polymerase (e.g., Promega PCR kit).

2 .. PCR reaction is done for 30 cycles (denaturation at 94°C for 2 min, annealing at 55-65°C for 1 min, and extension at noc for 1 min) and final extension at noc for 10 min.

3. peR products are checked by loading in 0.8% agarose gel at 120 V.

3.3. Preparation of Nondenaturing Polyacrylamide Gel (12-15%) for SSCP Analysis (see Notes 3-6, 8-10)

1. Clean the gel plates with detergent and rinse well with distilled water. 2. Wipe the plates with absolute ethanol. 3. Clean the spacers with absolute ethanol and place securely on the bottom plate. Secure

the plates properly with clamps.

SSCP: Limitations and Solutions 255

4. Clean the combs with absolute ethanol and keep it ready to be inserted between the two plates after pouring the gel mix.

5. Prepare the gel mix (12-15%) by addi.ng a premade solution of acrylamide and bis-acrylamide at a ratio of 29: 1, adding lOX TBE to make final concentration of IX, 200 ilL of 10% APS, and 50 ilL of TEMED.

6. Mix all the constituents properly and pour between the two plates (see Notes 3-5). 7. Leave the assembly undisturbed until the gel becomes polymerized. 8. Prepare 'the samples for loading by taking 5 ilL of PCR product, adding 4.5 ilL IX TBE,

and 0.5 ilL of loading dye. 9. Denature samples'at 95°C for 5 min and chill immediately on ice for 5 min prior to loading.

10. Load all. of the sample prepared into the appropriate wells of the non-denaturing gel, and electrophorese at a constant voltage at room temperature or at 4°C. (See Note 6.)

3.4. Silver Staining of SSCP Gel I. After completion of the gel run, fix the gel in fixative for about 30 min, 2. Stain the gel in 0.1 % silver nitrate solution for 15 min. 3. Wash the gel three times with autoclaved water. 4. Develop by adding 1.5% NaOH solution and wait till the bands appear. 5. Throw away the previous solution and add 0.75% sodium carbonate solution. 6. Photograph/scan the gels for record purposes. 7. For long-term storage: dry the gels in a gel dryer or store in a I % acetic acid solution. (See

Note 7.)

3.5. PAGE Elution of Bands Showing Variation (see Notes 2 and 3) I. Cut out the band showing variation, and mince it well with the help of a microtip in an

Eppendorf tube. 2. Add 400 ilL of PAGE elution buffer and incubate at 37°C overnight with shaking. 3. Centifuge at 16,OOOg 4°C for 1 min. 4. Take the supernutant and add IIlOth vol of sodium acetate, pH 5,2, followed by I mL

ethanol. 5. Keep at -80°C for I h. 6. Centrifuge at 16,000g 4°C for 20 min. 7. Wash twice with 70% ethanol, followed by centrifugation at 16,000g, 4°C for 10 min. 8. Air-dry DNA and dissolve in 10 ilL autoclaved and double-distilled water. 9. PCR-amplify the eluted fragment DNA.

3.6. Agarose Purification of DNA Bands I. Run the PCR product to be eluted en a 0.8% agarose gel. 2. Excise the PCR-amplified product from the agarose gel and put it in a 1.5-mL Eppendorftube. 3. Add I mL of saturated phenol, pH 8.0 into the tube, and freeze it at -80°C. Take out the

frozen tube, and thaw it completely. Freeze-thaw the tube three times. 4. In the final step, take out the frozen tube and centrifuge at a high speed of 13,OOOg for 20

min at room temperature. 5. Take out the aqueous phase and transfer to a fresh tube. Add an equal volume of chloro­

form into the tube, mix, and centrifuge again at 13,OOOg for 10 min. 6. Transfer the aqueous phase and add 11l0th vol of 3 M sodium acetate, pH 5.2, and make

up tol mL yol by absolute ethanol. Allow the DNA to precipitate for 30-45 min at -80°C. 7. Centrifuge at 16,000g at 4°C for 20 min for precipitating DNA.

256 Gupta et al.

8. Wash the DNA pellet with chilled 70% ethanol for 10 min at 4°C. 9. Dry the pellet and finally dissolve it in 10 J.lL of autoclaved distilled water.

10. Check the quality of DNA by running in an agarose gel and quantitate the amount for cloning.

3.7. Ligation of Gel-Eluted Product for Determination of Allele Sequence l. Mix 2X reaction buffer, T-tailed vector (e.g., pGEM-T Promega ligation kit), enzyme

(ligase), and DNA insert (gel-eluted product) in a O.S-mL Eppendorf tube. Mix well and make up the volume to 10 J.lL (per instructions given in, e.g., the Promega PCR ligation kit).

2. Incubate at 16°C for 4-6 h, followed by an overnight incubation at 4°C.

3.B: Transformation of Ligated Product and Screening of Positive Clones I. Carry out the transformation using competent cells (e.g., E.coli strains DHSa, XL-I). 2. Rescreen for positive clones through colony PCR using the same set of primers as was

used for amplifying genomic DNA and at the same PCR conditions. 3. Sequence the resulting PCR product manually, or by an automated sequencer.

1.9. Recombinant Plasmid Isolation From Positive Clone for Further Use as Control in SSCP Gels (see Note 3d)

1. Inoculate 5 mL of fresh LB medium with a single colony and grow it at 37°C overnight with high-speed shaking.

2. Isolate the plasmid as in the instructions of a plasmid isolation kit (e.g., Sigma or Promega plasmid isolation kit).

3. PCR-amplify the plasmid and run it for all subsequent g~ls of the same amplicon.

4. Notes 1. General considerations while doing PCR-SSCP:

a. Prepare and store the chemicals as mentioned, and handle with care. b. While isolating genomic DNA, handle with care to minimize the shearing of DNA. c. DNA is better purified with phenolJchloroform. d. For PCR reaction, use fresh sterile tips, tubes, and aseptic bench area. e .. Filter the gel mix before adding APS and TEMED. APS should be freshly prepared. f. Any indecision in band shifts can be removed by repeating the PCR either fresh or by

reamplifying the eluted variant band.

2. False bands seen: sometime:, SSCP results may be misleading, and false bands may appear:

a. At times, deformer bands appear in some lanes. Therefore, during sample prepara­tion, ensure an equal quantity of DNA and uniform treatment of all samples. In order to confirm that the extra bands appearing in some lanes are the variant bands, these should be cut out, eluted, amplified, and run again in an SSCP gel (see Note 3).

b. The presence of repetitive sequences causes primei slippage and hence the generation of extra bands during PCR amplification, a possible cause of false bands. To avoid this, try to keep PeR conditions as stringent as possible.

c. The Taq polymerase used for PCR amplification can also introduce mutations. It is preferable to use high-proofreading-activity polymerase rather than other polymerases with high fidelity. PCR-SSCP should be repeated for all the samples showing varia­tions, and their profiles should be confirmed before they are sequenced.

ssep: Limitations and Solutions 257

3. Appearance of extra bands in all the lanes: sometimes all the lanes show a number of bands, which makes the analysis difficult, as these may be confused with the mutant bands:

a. Nonspecific bands generated during PCR, which could be owing to nonstringent con­ditions or primer dimers. This can be controlled by maintaining stringent PCR condi­tions by reducing target or salt concentration, or keeping annealing temperature high. Also, the primer cOQcentration should be kept low. It is suggested to always check the PeR standardization results on acrylamitle gel along with marker. An agarose gel is not sensitive enough to discriminate bands differing by only a few base pairs.

b. Loading of high amounts of PCR products can also make visible the otherwise faint bands. Therefore, it would be good to optimize the amount of PCR sample to be used for loading depending on the PCR amplification, gel thickness, and well size. Some­times reducing the PeR amount can also reduce the intensity of the extra bands.

c. The PeR fragment adopts more than a single conformation or deformer. Adoption of more than one stable form is sequence-dependent, but varying physical conditions like pH, temperature, voltage, and so on may reduce the appearance of extra bands. This can also occur during temperature fluctuations. It is important to check for tem­perature fluctuations during the run. A deformer is less intense than the original band. Bands can be PAGE-eluted and PCR-amplified. Reamplified products should be elec­trophoresed along with the genomic control samples. A similar multiple band profile as earlier indicates the band is a deformer.

d. Double-stranded DNA, as seen in gels of higher concentration: run two to three con­trol samples for which the profile is known with the samples to be analyzed. Also, run a sample that has not been denatured to determine the location of double strands in the gel.

e. Partially denatured doublt>-stranded DNA takes a different conformation than single­stranded DNA and fully double-stranded DNA: denature the samples for at lea~t 5 min, d.ill immediately, and load immediately.

4. Gel polymerization problems: sometimes the gel does not polymerize well:

a. This could be caused by dirty plates or the use of chemicals of poor quality. It can be avoided by proper cleaning of plates using detergent, rinsing with autoclaved water followed by ethanol, wiping, and air-drying. Also, the gel solution should be made fresh and filtered, TEMED and A~S should be of good quality, APS should be made fresh, and its concentration should be checked; this can also be increased (under the optimal range).

b. Gel polymerization may take longer at low room temperatures. A table lamp or a heat convector should be used in such conditions.

5. Difficulty in resolving variant bands: sometimes variant bands do not resolve at all from the normal bands:

a. At times. three bands appear under heterozygous conditions instead of the expected four-band profile, in which two bands take a similar conformation and hence show an identical mobility. In such cases, it is not important to resolve the three-band profile into a four-band profile.

b. Sometimes the variant strand may not resolve from the normal band under a particular set of conditions, as both of them may have same mobility. In such cases, SSCP has to be standardized, as mentioned in Subheading 1.2.

c. An inadequate run of the sample also may not resolve the variant bands completely.

6. Problems in sample loading: sometimes improper loading leads to formation of a diffused band pattern:

258 Gupta et al.

a. The presence of mineral oil may interfere with loading. Try using no mineral oil while loading.

b. The sample may not settle in the well; use Ficoll or glycerol in the denaturing dye.

7. Silver staining problems: sometimes the gel may not stain well, or ghost bands may ap­pear in the middle section of the gel; instead of staining black, it remains clear and with­out stain:

a. Improper fixing and improper staining. Gels should be fixed properly, and freshly made silver nitrate and developer should oe used.

b. Bands are not clearly visible after staining if the amplification during peR is low. Therefore, peR conditions and the amount loaded should be standardized by check­ing the efficiency of peR amplification and its signal on agarose gel.

c. If the signal is good enough to be detected on PAGE, then the problem is in the stain­ing step. Reduce the number of intermediate washings. Shake the gel well, and mix all the solution with the gel for uniform staining.

8. Sample forms large streaks back toward the well:

a. This may be owing to the dry run of the gel. Try to avoid the dry run, which might allow evaporation of the buffer or some leakage. Seal the space between the plates and the tank to avoid leakage from the upper tank to the lower tank.

b. Try changing the gel percentage; an inappropriate percentage might lead to streak formation.

9. Uneven band patterns: sometimes bands are U-shaped, smeary, or diffused; they may show smiling or frowning:

a. The bottom of the well may not be flat. This could be ewing to improper polymeriza­tion of the gel. Take care during polymerization, as discussed in Note 1. Also, take out the combs only after the gel is properly polymerized. Always flush the well with buffer or water before loading in order to remove the residual gel solution, which might polymerize in the wells and lead to an uneven well bottom.

b. Uneven thickness of the gels owing to a lack of compatibility between combs and spacers causes film formation in the wells and hence improper loading and band pat­tern. Therefore, spacers and combs should be c; identical thickness to avoid uneven polymerization and film formation.

c. At times the appearance of bubbles in the gel matrix or at the interface of the gel and the buffer interfere with the current flow and affect the quai,ty of the run. The lanes which show the presence of air bubbles within the matrix could be avoided when running precious samples, and the bubbles at the interface could be removed by flush­ing with the help of a syringe and a needle.

d. This pattern may occur because the temperature is higher at the center than at the edges as joule heating effects are more easily dissipated at the edges of the gel; since electrophoretic mobilities vary inversely with the viscosity of the solvent, DNA samples in the center of the gel migrate faster than sample near the edges. Therefore, it is important to maintain the temperature of the matrix.

10. Gel-to-gel variations are seen even under :;imilar running conditions: sometimes two gels run at different times but in the similar conditions show varying profiles. Minor varia­tions in gel percentage, temperature, buffer concentration. pH. and so on can give a dif-

ssep: Limitations and Solutions 259

ferent profile for the same amplicon in two different gels~ The parameters influencing the outcome should be maintained as constant as possible. The following important points should be taken into account:

a. Genomic DNA should be quantitated carefully, and the amount used for PCR should be optimal.

b. The pH of the running buffer should not increase. The running buffer should not be reused again and again, although it can be used at least twice.

c. Try to maintain a constant temperature, voltage, and running time of the samples.

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260 Gupta et at.

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