Nov 25, 1991 - Ping Fu and Bronwyn Evans*. Howard Florey ... Marker; lane 2: 1st round PCR using HI specific A-chain primer (expected product size 4.2 kb) ...
.=) 1992 Oxford University Press
Nucleic Acids Research, Vol. 20, No.
11
2903
A novel PCR method for amplifying exons (or genes) over intragenic (or intergenic) regions in the genome Ping Fu and Bronwyn Evans* Howard Florey Institute of Experimental Physiology and Medicine, Parkville, Victoria 3052, Australia Submitted November 25, 1991 Amplification of two exons or genes across a large intragenic or intergenic region by PCR has been limited by the low yield of PCR products from long template sequences. Here we describe a two-stage PCR method which allows amplification of DNA from linked exons or genes in the genome. The procedure is based on the premise that although large PCR products are not detectable by hybridization, there will be a sufficient number of molecules to provide template for a second round of amplification. The protocol is shown in Fig. 1. Genomic DNA is amplified using two outside primers (a and b) corresponding to the 5' and 3' ends of exons (or closely linked genes) I and II respectively. After removing the free primers by spin filtration (2 times, centricon-I00), the PCR product is repaired by Klenow fragment and phosphorylated by kinase. The diluted DNA (< 2 Ag/ml) is circularized with T4 DNA ligase, and then amplified using two inside primers (c and d) corresponding to the 3' and 5' ends of exons (or genes) I and II, but having opposite orientation to the first-round primers. The shorter second-round PCR product can be analysed on an agarose gel and by Southern blotting. The procedure was tested using two human relaxin genes (HI and H2), which comprise exons I and II (encoding B and A chains respectively) interrupted by 3.7 kb introns (1). The first round of PCR was carried out using a 5' primer (a) common to both genes and 3' primers (b) specific for the A-chain of HI or H2. A second round of PCR was performed on the circularized firstround products using primers c and d (common to both HI and H2). Products of 264 bp were obtained after the second-round PCR (Fig. 2A, HI: lane 3; H2: lane 5). These were analysed by Southern blot using B-chain specific probes for H I or H2 (Fig. 2B or 2C). We have thus shown the linkage of the appropriate exons I and II in relaxin genes HI and H2 (1). This method is suitable for amplifying two exons separated by a large intron and for demonstrating the linkage of particular exons from members of a multigene family. It provides a faster alternative to the isolation of cDNA or genomic DNA clones from species where appropriate mRNA or libraries are unavailable. Although not tested here, the method should also allow an analysis of the linkage, order and orientation of closely linked genes.
ACKNOWLEDGEMENTS We thank G.Tregear, J.Haralambidis, P.Roche, R.Crawford, J.Gunnersen and D.Bowtell. This work was supported by the NH and MRC of Australia, the Ian Potter Foundation and the Myer Family Trusts. *
To whom correspondence should be addressed
REFERENCE 1. Hudson,P. et al. (1984) EMBO J. 3, 2333-2339.
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Figure 1. Two-stage PCR. Introns or intergenic regions are shown as straight lines. Filled boxes represent exons or genes I and II. Primers (a, b, c, d) are constructed to anneal to exons or genes with orientation shown by arrows. 1 2 3 4 5 6
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Figure 2. Analysis of PCR reactions. 1.5 ltg of human genomic DNA and 30 pmol of each primer were used in a reaction volume of 100 pl. (A) lane 1: MW Marker; lane 2: 1st round PCR using HI specific A-chain primer (expected product size 4.2 kb), PCR conditions: 94°C 3 min, 30 cycles of 94°C 1 min, 50°C 1 min, 72°C 3 min, and 72°C 10 min; lane 3: 2nd round PCR of lane 2 products; lane 4 and 5: as for lanes 2 and 3, except using H2 specific A-chain primer (expected product size 4.2 kb); lane 6: control (genomic DNA directly amplified using primers c and d). (B), (C) Southern blot analysis using internal oligonucleotide probes specific for the B-chain of HI (B) or H2 (C).
