Molecular Sex Determination Using LAMP-Based Isothermal Amplification
Introduction:
The determination of sex in human biological samples is a fundamental part of forensic casuistry, especially in those involving sexual assault. Commercial multiplex PCR-based kits that commonly include the amelogenin gene (AMEL) are commonly used, although inconsistencies have been reported in some populations due to deletions at this site.
Isothermal loop-mediated amplification (LAMP: Loop-Mediated DNA Amplification) is a versatile DNA amplification technique, with greater efficiency, specificity and yield compared to conventional PCR. The procedure itself is simple from the experimental point of view since it is an isothermal reaction mediated by Bst DNA polymerase that allows the continuous synthesis of DNA copies by self-displacement of the chain, which increases its sensitivity and prevents need for equipment that performs tempered cycles. Amplification products can be detected by agarose electrophoresis, turbidity analysis, as well as by simple vision using pH-sensitive dyes.
The present proposal is based on the evaluation of a multiplex LAMP protocol for the detection of different specific, unusual markers (TSPY3, TTTY2, TTTY7) of the human Y chromosome in combination with the first intronic region of the AMEL gene. Our system uses a set of 6-8 primers for each target, designed to improve sensitivity and specificity, reducing detection time to just 45 minutes.
Controlled mixtures of male and female human DNA simulating forensic samples will be tested at different ratios, as well as sensitivity levels at male DNA dilutions and specificity against domestic species commonly found in forensic contexts.
Goals:
1.- Disseminate the use and various applications of the LAMP technique.
2.- Evaluate the performance of the LAMP method for the molecular determination of sex in humans, specificity and sensitivity limits including mixtures, with controlled samples that simulate a forensic context.
3.- Gather ideas about the potential application of the LAMP technique from different laboratories with proven forensic expertise.
Methodology:
Interested laboratories can register their interest in participating by filling out their data and authorization to be contacted by the exercise coordinators in the following online form:
Participating laboratories will receive from us sufficient primer mix and blind samples for the different contexts (mixes, male DNA dilution, domestic animal DNA).
New England Biolabs will provide the colorimetric 2X WarmStart LAMP Mix to carry out at least 50 reactions (15 μl), enough to evaluate each item in triplicate, and even with a certain excess to evaluate own reference samples. The final result of the reactions must be recorded photographically, and the results format that will be designed for this purpose must be filled out.
If additional information or clarification of doubts is needed regarding the experimental protocol, Germán Burgos should be contacted via email (see end of document).
The coordinators will compile the results and issue a preliminary report to communicate the set of findings during the GHEP-ISFG 2023 meeting. Once the results have been discussed, the final version of the relevant scientific communication will be drafted.
Requirements for participants:
• Be members of the GHEP-ISFG and be up to date with the membership fees.
• A participation fee of 50 euros must be paid within the established period.
Deadlines
Registration deadline: November 30, 2022
Payment deadline: February 7, 2023
Deadline for sending the results: June 28, 2023
Presentation of results: July 26 to 28, 2023 during the XXVIII GHEP-ISFG Meeting (Cartagena-Colombia)
Notes:
New England Biolabs, manufacturer of the LAMP colorimetric mix supports this initiative by using its distribution channels in various countries to provide the mix free of charge, even for 30 participating laboratories.
The photographic records of the reactions, spatially distributed according to a standard template, and possibly the records of the 2% agarose electrophoresis (for those participants who decide to perform them) must be sent as evidence of the results and/or to resolve any doubts that may arise.
For the publication of results, a limit of one author is established for each registered laboratory, which will be classified in alphabetical order, including the organizers.
Coordinators and contacts:
German Burgos Figueroa, University of the Americas, Quito, Ecuador: hgermanburgosf@yahoo.com, german.burgos@udla.edu.ec
Leonor Gusmão, UERJ, Rio de Janeiro, Brazil: leonorbgusmao@gmail.com
Jacobus de Ward, University of the Americas, Quito, Ecuador: jacobusdeward@gmail.com
To learn more….
We share the following link, the proceeding that we have published in FSIGSS, where we describe our results in detail, for additional information contact GBurgos via email.
https://www.sciencedirect.com/science/article/abs/pii/S1875176822000853
LAMP video tutorial: https://international.neb.com/tools-and-resources/video-library/loop-mediated-isothermal-amplification-lamp-tutorial?autoplay=1 © Copyright 2022 New England Biolabs. All Rights Reserved.