DEVELOPMENT OF PCR-SSCP SILVER STAINING TECHNIQUE FOR SCREENING OF THE C7P7 7 MUTATIONS AND POLYMORPHISMS

3.2.1 Introduction

PCR-SSCP with silver staining provides another means of detecting DNA fragments and has the advantage of being non-radioactive and therefore safer to use with a reasonably long shelf life. In this study, the sensitivity of silver staining for detection of SSCP variants was compared with the radioactive technique using the conditions described in the methods. The nine sequence variations in the coding region of the CYP 17 as described in Chapter 3.1 were included in this part of the study. Eight of these were single base pair changes, and with one exception, heterozygous. The ninth mutation was a 5 bp deletion on a single allele. Details of all the sequence variations used in this part of this study and the results of screening by the radioactive SSCP techniques are summarized in Table 8.

The CYP 17 genes from leucocyte DNA of four 17-hydroxylase deficient patients were amplified exon by exon using identical sets of primers for both techniques as previously described. PCR products larger than 300 base pairs were digested with suitable enzymes to generate fragments of less than 200 base pairs before electrophoresis. Normal DNA samples were run at the same time to serve as controls. Twelve gel conditions were used to run the PCR products: 6%, 8% or 10% PAGE with or without 5% glycerol at room

Base change and effect Exon Patient Zygosity Result of radioactive SSCP CAT46CAC (silent,His) 1 3 Heterozygous Positive TCT65TCG (silent,Ser) 1 1,3,4 Heterozygous Positive CGG96TGG (Arg->Trp) 1 2 Homozygous Positive GAT139GAC (silent,Asp) 2 2 Heterozygous Positive ACC131TCC (T hr^S er) 2 3 Heterozygous Positive GAG194TAG (Glu-^STOP) 3 1 Heterozygous Negative CGA239TGA (A rg^STO P) 4 1 Heterozygous Negative GAT280GAC (silent,Asp) 5 4 Heterozygous Positive deletion of TGGAC (stop 387) 7 3 Heterozygous Positive

Table 8. Nine sequences variations and the results of the radioactive SSCP technique used

in this study.

Positive = Different SSCP patterns from normal Negative = The same SSCP patterns as normal

3.2.2 Results

After completing all twelve gel conditions for the silver staining technique, all the mutations/polymorphisms in the coding region of the CYP 17 which had been detected using radioactive SSCP in this study yielded positive results. The positive control, a compound heterozygotes for mutants in exons 3 and 4 which failed to show different SSCP patterns using the radioactive technique under three gel conditions (Figure 14) was again negative. However, digestion of exons 3 and 4 with the enzyme Sau 3A1 to cut each exon into two asymmetrical fragments yielded positive screening results compared with the normal sequences on the PCR-SSCP silver staining method at both room temperature condition and 4°C (Figure 15a&b). The PCR fragment was digested with Sau 3A1. The fragments which contained a sequence change was 175 and 169 base pairs for exons 3&4 respectively. The results of gel conditions and screening patterns are summarized in Table 9.

One pathological mutation in exon 2 and one silent mutation in exon 2 which gave the same variant SSCP patterns on all three gel types by the radioactive technique (Figure 9) showed distinguishable variant band shifts from the normal control and also different patterns from each other under six gel conditions when electrophoresis was performed at room temperature only (Figure 16). Under only two gel conditions, both fragments gave identical variant SSCP patterns from normal. The gels conditions and the results of the screening for these mutations are shown in Table 10.

Three pathological mutations in exon 1, exon 2 and 7 were clearly detected by both the radioactive and the silver staining methods. For the silver staining technique.

electrophoresis atoom temperature only was adequate to give positive results, (detailed data and photographs are not shown)

Two common polymorphisms in exonl (Patient 1,3,4) gave different band shifts for both techniques. Two previously undescribed polymorphisms; one in exon 2 (Patient 2) which gave variant band shifts by the radioactive technique and the other in exon 5 (Patient 4) which gave barely perceptible differences by the radioactive method, could all be clearly distinguished from the normal by using the silver staining technique.

No pathological mutation was identified in two patients (patients 4&5). One of the two individuals had a neutral mutation in exon 5 and two intronic changes in intron 3 and 4 (see 3.1.3.2 and 3.1.5). Samples from these two patients were also included for comparison while this study was performed. The two samples gave different SSCP patterns for exon 4 (Figure 17). Patient 4 was homozygous for the change at the 5' end of intron 4 and both parents were heterozygous and homozygous for this variant confirmed by direct sequencing. Thus, Patient 5 was assumed to inherit the same alleles as patient 4 and was therefore expected to yield a different SSCP pattern from normal but failed to show a different band shift.

Exon3 (digested) Exon4 (digested)

Room temp 4°C Room temp 4°C

6%PAGE,G - +/- - - 6%PAGE,N - - + - 8%PAGE,G + + - - 8%PAGE,N - +/- - - 10%PAGE,G - - - - 10%PAGE,N - +/- + +

Table 9. PAGE-silver staining SSCP analysis of exon3 and exon 4 containing mutation G>T and C>T which failed to show different band shift for the radioactive PCR-SSCP technique.

+ = Different band shift and/or extra band +/- = Doubtful band shift and/ or faint extra band - = Normal pattern

N = Without 5% glycerol G = With 5% glycerol

Patient!, exon! Patient 3, exon! GAT139GAC ACC131TCC (silent,Asp) (T hr^Ser) 6%PAGB,N - - 6%PAGE,G +* +* 8%PAGE,N 4- + 8%PAGE,G - - 10%PAGE,N + + 10%PAGE,G + +

Table 10. Comparison of the PAGE-silver staining SSCP patterns of exon 2s containing two different mutations.

= Both mutations gave the same SSCP patterns as normal

+ = Both mutations gave variant SSCP patterns from normal and could be distinguished from the other

+* = Both mutations gave the identical variant SSCP patterns from normal

N= Without 5% glycerol G= With 5% glycerol

3.2.3 Additional study of the effect of decreasing PCR fragment size and the sensitivity of silver staining method

The initial PCR products obtained from the amplification of exons 3 and 4 of the control sample were 323 and 315 base pairs in length respectively. The silver staining gel system detected both sequence variants of the digested PCR fragments in this sample while the radioactive technique failed to give positive results. To see the effect of varying fragment size on detection of the same single base change, new primers were designed to cover the whole exon area only in both exons 3 and 4, yielding smaller PCR fragments of 270 and 230 base pairs respectively. Analysis revealed a mobility shift in the smaller fragment of exon 4 compared with the same size fragment from a normal allele. No mobility shift was observed with the larger fragment of exon 3. Summarized results of this additional study are shown in Table 11.

Exon 3 (undigested) Exon 4 (undigested)

Room temp 4°C Room temp 4°C

6%PAGE,G - - + + 6%PAGE,N - - + + 8%PAGE,G - - + - 8%PAGE,N - - + - 10%PAGE,G - - + - 10%PAGE,N - - + -

Table 11 .PAGE-silver staining SSCP analysis of PCR products from new primers of exon 3 and exon 4 of the control sample.

+ = Different band shift N = Without 5% glycerol

6%PAGE 6%PAGE 6%PAGE