Counting CAG repeats in the Huntington's disease gene by restriction endonuclease EcoP15I cleavage

Abstract

Huntington's disease (HD) is a progressive neurodegenerative disorder with autosomal-dominant inheritance. The disease is caused by a CAG trinucleotide repeat expansion located in the first exon of the HD gene. The CAG repeat is highly polymorphic and varies from 6 to 37 repeats on chromosomes of unaffected individuals and from more than 30 to 180 repeats on chromosomes of HD patients. In this study, we show that the number of CAG repeats in the HD gene can be determined by restriction of the DNA with the endonuclease EcoP15I and subsequent analysis of the restriction fragment pattern by electrophoresis through non-denaturing polyacrylamide gels using the ALFexpress DNA Analysis System. CAG repeat numbers in the normal (30 and 35 repeats) as well as in the pathological range (81 repeats) could be accurately counted using this assay. Our results suggest that this high-resolution method can be used for the exact length determination of CAG repeats in HD genes as well as in genes affected in related CAG repeat disorders.

Figure 1

EcoP15I cleavage of DNA substrates that contain CAG repeats of different lengths. (A) Recognition and cleavage sites of restriction endonuclease EcoP15I in the DNA molecule. For DNA restriction, the enzyme needs two 5′-CAGCAG sequences being inversely oriented in the double-stranded DNA. Cleavage occurs 25–27 bp downstream of one of the two inverted sites (see Introduction). (B) General chart of the used DNA substrates that contain a number of n CAG repeats and an additional inverse EcoP15I recognition site 5′-CTGCTG at a distance of 72 bp from the 3′-end of the CAG repeats. Two CAG trinucleotides correspond to one EcoP15I recognition site 5′-CAGCAG. Thus, an expansion of n CAG trinucleotides results in a series of (n-1) EcoP15I recognition sites that overlap by 3 bp. Arrows indicate the EcoP15I cleavage sites 25–27 bp downstream of the various recognition sites. In the lower part of the chart the fragment patterns expected from cleavage either at one of the CAG repeats or at the inverse EcoP15I recognition site ‘i’ are schematically shown. (C) Calculated lengths of the DNA fragments expected from EcoP15I cleavage of the DNA substrates fCAG30, fCAG35 and fCAG81. Fragment lengths are assigned to the corresponding EcoP15I recognition sites nos 1 to (n-1) within the CAG repeats or to the inverse site ‘i’. (D) EcoP15I cleavage patterns of the DNA substrates fCAG30, fCAG35 and fCAG81. The DNA substrates were radioactively labeled at both ends. Each substrate (50 fmol) was incubated in the presence or absence of a 10-fold molar excess of EcoP15I enzyme over DNA substrate at 37°C for 30 min. Cleavage was analyzed on a non-denaturing 5% (w/v) polyacrylamide gel as described in the Materials and Methods. The ladders of EcoP15I cleavage fragments were marked by brackets. M, molecular weight marker; uc, uncleaved DNA substrates; i (5′), 5′-terminal DNA fragment after EcoP15I cleavage at the inverse EcoP15I recognition site; i (3′), 3′-terminal DNA fragment after EcoP15I cleavage at the inverse EcoP15I recognition site; *, DNA fragments generated by EcoP15I cleavage at EcoP15I recognition sites nos 1 to (n-1) within the CAG repeats.

Figure 2

Influence of EcoP15I concentration on cleavage efficiency and cleavage pattern. The ³²P-labeled DNA fragments fCAG30 (A), fCAG35 (B) or fCAG81 (C) were cleaved with increasing amounts of EcoP15I (for cleavage conditions and analysis see Fig. 1D). Lane 1, without EcoP15I; lanes 2–4, molar ratios of EcoP15I enzyme to DNA substrate of 1, 10 or 20. For abbreviations see legend to Figure 1.

Figure 3

Analysis of the EcoP15I cleavage pattern of fCAG30 using the ALFexpress DNA Analysis System. DNA substrate fCAG30 was Cy5-labeled either (A) at the 5′-end or (B) at the 3′-end and incubated with EcoP15I at molar ratios of enzyme [E] to DNA substrate [S] of 1, 10 or 20. Cleavage products were separated on non-denaturing 7% (w/v) polyacrylamide gels and analyzed in the ALFexpress DNA Analysis System. Output data show DNA fragments as fluorescence intensity F in arbitrary units. The upper panels show all cleavage products and uncleaved DNA substrate. The lower panels are a close up of the cleavage products. For abbreviations see legend to Figure 1. The molecular weight marker fragments contained two HpaII ends and were therefore Cy5-labeled at both ends. In contrast, fCAG30 was labeled at only one end. As Cy5 is known to change the electrophoretic mobility of DNA fragments (42), the marker could not be used as an absolute but as a relative length standard.

Figure 4

Counting of CAG repeats in fCAG30, fCAG35 and fCAG81 at an equimolar enzyme to substrate ratio. The DNA substrates fCAG30, fCAG35 and fCAG80 were Cy5-labeled at their 3′-ends. EcoP15I cleavage fragments were separated on non-denaturing 7% (w/v) polyacrylamide gels and analyzed using the ALFexpress DNA Analysis System. Output data show DNA fragments as fluorescence intensity F in arbitrary units. Cleavage by EcoP15I resulted in a ladder of DNA fragments in 3 bp steps from 139 to 223 bp for fCAG30, 139 to 238 bp for fCAG35 and 139 to 376 bp for fCAG81. In addition, a 117 bp DNA fragment was observed due to the cleavage at the inverse EcoP15I recognition site ‘i(3′)’.