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Recombinase polymerase amplification for human male determination from semen |
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| รหัสดีโอไอ | |
| Creator | Monthira Monthatong |
| Title | Recombinase polymerase amplification for human male determination from semen |
| Contributor | Anupong Sukjai |
| Publisher | Asia-Pacific Journal of Science and Technology |
| Publication Year | 2566 |
| Journal Title | Asia-Pacific Journal of Science and Technology |
| Journal Vol. | 28 |
| Journal No. | 2 |
| Page no. | 8 |
| Keyword | Male-specific marker, RPA, SRY1.Introduction Biological evidence can be used to determine the gender of victims or suspects in forensic cases, including murder, rape and missing persons [1]. Commonly, the morphological and anatomical characteristics of sex organs are used to determine the gender of an unidentified body. Currently, the polymerase chain reaction (PCR) or multiplex PCR techniques are routinely used in the molecular biological process for determining the human gender of organic traces [2]. In forensic study, gender determination was commonly performed using PCR with both the amelogenin (AMEL) and sex-determining region Y (SRY) genes [3]. However, PCR-based assay still has some disadvantages, such as being time-consuming, depending on complicated thermal cycling machines, having a high energy cost, and requiring precise laboratory facilities and highly skilled operators, which restrict the application of PCR in small or unspecialized laboratories and in resource-limited settings. These limitations in PCR-based methods have urged the development of a new molecular biological technique known as isothermal Deoxyribonucleic acid (DNA) amplification that has been extensively reviewed [4-9]. One isothermal DNA amplification technique is called recombinase polymerase amplification (RPA) [10]. RPA is a highly sensitive and selective isothermal amplification technique, performed at a single temperature in the range 37-42?C with minimal sample preparation and is capable of amplifying as low as 1-10 targeted DNA copies in less than 20 min without the need for an initial denaturation step or the use of multiple primers. RPA uses three proteins, including recombinase, single-stranded DNA binding protein (SSB) and strand-displacing polymerase, to amplify double-stranded Deoxyribonucleic acid (dsDNA) in a similar way to the PCR method [11,12]. In the RPA reaction, the recombinases combine with the primers to form recombinase-primer complexes that subsequently scan the double-stranded DNA template for the homologous sequence. The strand displacement reaction is initiated to synthesize new DNA sequences when the primers hybridize to their complementary strand. The displaced template strand is stabilized by SSB and the recombinase dissociates and allows elongation of primers by strand-displacing DNA polymerase. The process generates a complete copy of the |
| URL Website | https://www.tci-thaijo.org/index.php/APST |
| Website title | https://so01.tci-thaijo.org/index.php/APST/article/view/264434 |
| ISSN | 2539-6293 |
| Abstract | Recombinase polymerase amplification (RPA) is a simple, rapid, highly effective, specific, and sensitive technique used for amplification of specific DNA regions at a constant, low temperature (between 37 ?C and 42 ?C) without using a thermal cycler. The RPA products can be detected using agarose gel electrophoresis. This research developed primers specific to the SRY (sex-determining region Y) gene in RPA assay. RPA primers were designed using parameters according to the instruction manual (TwistDx?) and Primer-BLAST software based on the human SRY gene (accession number: JQ811934.1) obtained from the GenBank database. The designed primers were further tested using RPA at 39 ?C for 20 min. The results revealed that the set of developed RPA primers could detect all male DNA from human semen samples. Sensitivity analysis showed that the detection limit was determined to be 0.01 ng for template DNA. Therefore, the RPA primers designed in this research are an alternative valuable tool for sex determination as a part of biological evidence in forensic casework. |
