Linked linear amplification: a new method for the amplification of DNA.

BACKGROUND: Linked Linear Amplification (LLA) is a new nucleic acid amplification method that uses multiple cycles of primer extension reactions. The presence of nonreplicable elements in LLA primers renders primer extension products unusable as templates for further amplification, leading to linear accumulation of products. Through the use of nested primers, linear reactions can be "linked", providing total amplification yields comparable to those obtained by PCR. METHODS: The LLA model predicts (a) that amplification yield will approach that of PCR as the number of primers increases and (b) that the unique composition of LLA products will give lower carryover amplification efficiency compared with PCR. To test these hypotheses, the human ss-globin gene was amplified by 10-, 14-, or 18-primer LLA and the yield was compared with PCR. Carryover contamination was simulated by reamplifying a dilution series of LLA or PCR products. To demonstrate the clinical utility of the method, LLA coupled with allele-specific oligonucleotide (ASO) capture was used to detect the factor V Leiden mutation in a panel of 111 DNA samples. RESULTS: Fourteen- and 18-primer LLA gave amplification yields comparable to PCR. However, LLA carryover amplification efficiency was four orders of magnitude lower than that of PCR. The LLA-ASO assay detected the correct factor V Leiden genotype in all 111 samples. CONCLUSIONS: LLA is a robust target amplification method that is comparable to PCR in yield. However, LLA is more resistant to false results caused by carryover amplicon contamination.

Evaluation of the BeTha gene 1 kit for the qualitative detection of the eight most common Mediterranean beta-thalassemia mutations.

We describe the evaluation of the Bio-Rad BeTha Gene 1 kit (Bio-Rad Laboratories, Hercules, CA), a DNA-probe assay designed for the qualitative determination of the eight most common Mediterranean beta-thalassemia mutations. The kit utilizes the principle of allele-specific oligonucleotide (ASO) hybridization. Following sample preparation and in vitro DNA amplification by the polymerase chain reaction (PCR), an allele-specific detection of the amplified products by a nonradioactive enzymatic assay is performed. Genomic DNA is prepared from an individual's whole blood with a DNA purification matrix. In a second step, the beta-globin gene is amplified in a multiplex PCR reaction containing four 5' biotinylated oligonucleotide primers. In a final step, an aliquot of the PCR reaction is first chemically denatured and then captured in two eight-well strips of a 96-well enzyme-linked immunosorbent assay (ELISA) plate by hybridization to an immobilized ASO probe. Each DNA sequence at each of the eight mutation sites is represented by one normal and one mutant ASO. During this capture/hybridization step, which is performed at 37 degrees C, only perfectly matched PCR products will be captured by an ASO. Subsequently, the allele-specific captured biotin-labeled PCR products are detected by a colorimetric enzymatic reaction. The system permits the detection of 16 beta-thalassemia alleles using a high-throughput format that can be automated easily. A clinical feasibility study was performed to evaluate the functionality (method comparison study, assay validity using samples previously collected and stored at various temperatures for different periods of time, interference on kit performance, and assay validity for prenatal diagnosis) and the usability (ease of use, sample throughput) of the kit. The analysis of 110 samples previously studied with reference methods showed 100% clinical sensitivity and specificity. We demonstrate here that the procedure not only increases the throughput of beta-thalassemia allele genotyping but also provides an accurate, rapid, reliable, and nonisotopic diagnostic tool.