The development of miniSTRs as a method for high-speed direct PCR
| Autor: | Roberta Fogliatto Mariot, Bruce McCord, Dide Boelens, Mirna Ghemrawi, Ate D. Kloosterman |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Combined DNA Index System
DNA polymerase Clinical Biochemistry 02 engineering and technology Computational biology 01 natural sciences Biochemistry Polymerase Chain Reaction Analytical Chemistry law.invention chemistry.chemical_compound law Genotyping Polymerase chain reaction biology 010401 analytical chemistry DNA Amplicon 021001 nanoscience & nanotechnology DNA Fingerprinting 0104 chemical sciences chemistry biology.protein Amelogenin Primer (molecular biology) 0210 nano-technology Nucleic Acid Amplification Techniques Microsatellite Repeats |
| Zdroj: | Electrophoresis6 References. 42(12-13) |
| ISSN: | 1522-2683 |
| Popis: | There are situations in which it would be very valuable to have a DNA profile within a short time; for example, in mass disasters or airport security. In previous work, we have promoted reduced size STR amplicons for the analysis of degraded DNA. We also noticed that shorter amplicons are more robust during amplification, making them inhibition resistant, and potentially applicable to high-speed direct PCR. Here, we describe a set of miniSTRs capable of rapid direct PCR amplification. The selected markers are a subset of the Combined DNA Index System (CODIS) loci modified to permit high-speed amplification. Using the proposed protocol, the amplification of eight loci plus amelogenin directly from a saliva sample can be completed in 7 min and 38 s using a two-step PCR with 30 cycles of 98°C for 2 s and 62°C for 7 s on a Streck Philisa thermocycler. Selection of DNA polymerase, optimization of the two-step PCR cycling conditions, the primer concentrations, and the dilution of saliva is described. This method shows great potential as a quick screening method to obtain a presumptive DNA profile when time is limited, particularly when combined with high-speed separation and detection methods. |
| Databáze: | OpenAIRE |
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