don’tt hrowth at away! -...
TRANSCRIPT
Summer Meeting May 30-31, San Antonio
Don’t Throw That Away! Get a Bigger Boost
Karl Reich
Introduction
Fluorescent Detection of Sperm from Sexual Assault evidence SPERM HY-LITERTM SPERM HY-LITERTM EXPRESS SPERM HY-LITERTM PI
Lateral Flow Immunochromatographic Strip Tests
RSIDTM-SALIVA RSIDTM-SEMEN
RSIDTM-BLOOD RSIDTM-URINE
Laboratory: Private DNA testing facility, suburban Chicago, ISO-17025, AABB, CLIA, NY-DOH
Newest Product: Post-PCR purification: Amplicon RxTM
The Problem
Partial DNA Profiles Insufficient amount of DNA
PCR inhibition
Poor quality DNA
Mutations at primer binding site
Analyzing only 4% of the reaction
Increase the amount of DNA, if possible
Increase the amount of DNA (repair?)
Use another kit (if possible)
Use all of the PCR reaction: Amplicon Rx
Purify the DNA, re-amp
Reminder
DNA Multiplex PCR Reactions Full size Rxn: 25 µL (50 µL)
Aliquot into Formamide: 1-1.5 µL (2-6%) of total PCR rxn
Left behind: 94-98% of the results!
No other analytical technique discards > 90% of the analyte! Not GC, not MS, not AA, . . . . .
Reminder
Electrokinetic Injection
Charged species enter the capillary under applied voltage
Amplicon amplitude (RFU) not linear with injection time
Complex relationship of voltage, current, time, charged species, . . .
Competition by salts, unincorporated oligonucleotides, nucleosides, . .
Amplicon Rx™
What is it: Post-PCR purification method for multiplex DNA-STR reactions (not for DNA sequencing rxns)
What does it do: Increases the RFU signal from PCR amplicons
How does it do it: Removes nucleotides, salts, and the bulk of the non-incorporated oligonucleotide PCR primers
What it does not do: It does not amplify, skew, alter or in any way change the PCR reaction
Amplicon Rx™ Procedure
load on column
Spin 3 min [Discard run through]
+ Formamide + Liz
[Incubate 5 min, RToC]
Spin 2 min
Load Elution on CE
+ Binding Buffer PCR Reaction
Amplicon Rx™ Results
After Amplicon Rx™
Touch DNA Samples (Identifiler)
Before
Amplicon Rx™ Results
PowerPlex16
Before & After Amplicon Rx™
Amplicon Rx™ Results
Before
After Amplicon Rx™
Amplicon Rx™ Results
Tested: ABI kits Promega kits Y-STR kits Mixtures Standards Full-sized PCR reactions (9->20x) Half-sized PCR reaction (7->10x) Quarter-sized PCR reactions (3-4x) . . . . .
Observed RFU Boosts: dependent on PCR reaction size, up to >20X!
Contact Information
Acknowledgments: Dr. Alex Sinelnikov (primary author) [email protected]
Sales & Support: Ms Dina Mattes [email protected]