CCR: a rapid and simple approach for mutation detection
CCR: a rapid and simple approach for mutation detectionWanli Bi and Peter J. Stambrook*
Department of Cell Biology, Neurobiology and Anatomy, University of Cincinnati, College of Medicine, Cincinnati, OH 45267-0521, USA
Received February 24, 1997;Revised and Accepted May 21, 1997
ABSTRACT
We describe a simple approach for detecting known mutations in genomic DNA. The strategy entails a DNA amplification reaction that combines the use of thermostable DNA polymerase and ligase, and that has been designated the Combined Chain Reaction (CCR). CCR consists of four phases: denaturation, annealing, elongation and ligation. Unlike most PCR-based mutation detection systems it relies on mismatch between primer and template at the primer 5' ends. It is rapid and simple, and requires neither the use of radioactivity, nor polyacrylamide gel electrophoresis, nor autoradiography for mutation detection at the single base-pair level.
The polymerase chain reaction (PCR) allows the amplification of a discrete segment of DNA from a large pool of irrelevant DNA sequence (1 ). Among its first applications was the detection of the known nucleotide substitution associated with sickle cell anemia (2 ). Since that time, PCR has revolutionized and become an essential tool for molecular biology. It is now used for diagnosis of hereditary and infectious diseases, routine DNA amplification, site-directed mutagenesis, cloning of genomic DNAs and cDNAs, DNA sequencing, DNA fingerprinting and forensic science. The Ligase Chain Reaction (LCR), was developed as an alternative amplification procedure and has been used for detecting known point mutations (3 ,4 ) and for introducing mutations into DNA in vitro. To combine the applications of PCR and LCR, we describe a DNA amplification strategy that utilizes both thermostable DNA polymerase and thermostable ligase in a single reaction. The designation for this combined chain reaction is CCR. Each CCR cycle consists of four steps: denaturation of template DNA, annealing of primers to single-stranded templates, elongation of primers by DNA polymerase, and ligation of the 3' OH end of an extended primer with the 5'-phosphate end of a downstream primer.
The underlying principle of the CCR approach is illustrated in Figure 1 . Like LCR, CCR requires four primers for mutation detection, but the primers are positioned differently. Two external primers flank the overall sequence that is amplified while two internal diagnostic primers have their 5' ends positioned at the putative mutation site. The differential ability to form a phosphodiester bond between perfectly matched ends and mismatched ends is the basis of LCR, and is also the basis for CCR in this application and is described in detail in the legend to Figure 1 .
To test the applicability and efficiency of CCR for detecting known mutations in genomic DNA, a known mutation at P53 in HaCaT cells was analyzed. The HaCaT cell line is derived from a human squamous cell carcinoma (5 ), and is a compound heterozygote at P53 with two different mutant P53 alleles (6 ). The mutation targeted for detection was a dinucleotide substitution CC -> TT at codon 281-282 in exon 8. Genomic DNA from the human fibrosarcoma cell line HT1080 was used as a control since this cell line lacks mutations in P53 within exons 5-9 (7 ). The external primer EP1 was positioned at nt 14 106-14 125 and the other external primer EP2 was placed at nt 14 804-14 785 on the other strand (Fig. 2 a). The internal primers extended from nt 14 512 to 14 535 and from 14 513 to 14 489. The internal primers MU and ML match the mutant P53 sequence; whereas the internal primers WU and WL match the wild-type P53 sequence. A third primer pair, CU and CL, that matched neither, was used as a control. When BCR was applied to HT1080 genomic DNA, the wild-type internal primers produced the predicted 699 bp fragment in addition to the two smaller 293 and 408 bp fragments (Fig. 2 b). The internal, mutant primers produced only the smaller fragments since the mismatched 5' ends are not ligated with sufficient efficiency to produce the 699 bp fragment. It is highly improbable that the two small fragments will be ligated in subsequent cycles because the 5' terminal nucleotides of each pair of diagnostic primers are complementary. In preceding cycles, an extra 2 nt will have been generated at the junction site further reducing the likelihood of ligation. This reduces background when diagnostic primers are mismatched with template. In contrast to HT1080 cells, HaCaT cells have one allele mutated at that site. Thus, the diagnostic, full length fragment was produced using either the MU/ML or WU/WL primer pairs, but not using the control primer pairs.
REFERENCES
1 Mullis,K., Faloona,F., Scharf,S., Saiki,R., Horn,G. and Erlich,H. (1986) Cold Spring Harbor Symp. Quant. Biol., 51,263-273.MEDLINE Abstract
