Dilution

Right: wikimedia.org

User:
Staci Bennett, Minnesota Bureau of Criminal Apprehension, St. Paul

Project:
Determining "whodunit," based on DNA evidence.

Problem:
It's sometimes difficult to isolate DNA amenable to PCR from stains in dark denim.

Solution:
Forensic labs do everything by the book. They follow an exhaustively validated and detailed standard operating procedure (SOP) in order for evidence to be admissible in court. The SOP even includes directions on how much wiggle room Bureau scientists have when diluting a sample; such variation is often needed to overcome PCR inhibitors.

For saliva or blood stains on clothing, for example, the SOP calls for a 3 x 3 mm denim cutting to be placed in extraction buffer with Proteinase K, vortexed, incubated at 56°C, and precipitated with phenol-chloroform. Yet denims are not all dyed the same way, and some dyes remain in the aqueous phase with the DNA, while others rinse away. "Depending on what phase the color will end up will give some indication of whether or not we'll have a problem," Bennett explains.

Bureau scientists do RT-PCR quantification using ABI's Quantifiler kits. The quantification process provides an indicator that allows them to see if an inhibitor is in the sample. Usually, diluting the sample — using 600 picograms per reaction instead of 1 nanogram — will be enough to get the PCR to work. If not, Bennett says, sometimes reamplifying with BSA and extra Taq will be effective.

Cost:
All told, Bennett spends about $50 per sample (or "profile"). This includes about $2 for Applied Biosystems' Quantifiler and $20 for ABI's Identifiler.