DNA Quantification

Why Quantify DNA?

All sources of DNA are extracted and are present in the samples to be examined

Assurance standards require human - specific quantification

Determine the appropriate amount of DNA template to include in future tests (e.g. PCR)

How much input human DNA is optimal with current commercial STR kits?

0.5 to 2.0 ng

Normalization

if the amount of DNA in a sample is outside of the target range for creating a ‘just right’ DNA profile, then the DNA amount must be adjusted prior to putting it into the PCR reaction

the process of achieving a DNA concentration that fits the optimal window for analysis is called normalization

this involves diluting the sample down to the desired range or concentrating it by removing excess fluid

UV Absorbance and Yield Gels limitations

these approaches are not sensitive they consume valuable forensic specimens that are irreplaceable

Slot Blot

Specific for human and other primate DNA

PicoGreen Microtiter Plate Assay

is capable of detecting as little as 250 pg of double - stranded DNA in a 96 well plate

is a fluorescent intercalating dye who fluorescene is greatly enhanced when bound to double stranded DNA

Quantifies total DNA and is not specific for human DNA

can be automated

What are Intercalating Dyes?

chemicals that bind between DNA base pairs, allowing detection in real-time PCR (SYBR green)

cost effective and simple and quantify low or degraded DNA

non-specific bind (e.g. primer-dimers) and requires optimization

AluQuant Human DNA Quantitation System

can be automated

End - Point PCR

less fancy and less expensive than qPCR

A single STR locus is amplified with DNA samples of known concentrations

a fluorescent intercalating dye such as SYBR Green can be used to detect generated PCR products

the level of DNA can be adjusted prior to amplifying the multiplex set of DNA markers in order to obtain the optimal results

monitors the level of PCR inhibitors

Real Time Quantitative PCR (qPCR)

the primary purpose in performing a DNA quantitation test is to determine the amount ‘amplifiable’ DNA

a PCR amplification reaction may fail due to the presence of coextracted inhibitors, highly degraded DNA, insufficient DNA quantity, or combination of all these

thus a test that can be accurately reflect both the quality and the quantity of DNA in a sample

qPCR Instrumentation

analyzes the cycle-to-cycle change in fluorescence signal resulting from the amplification of the target DNA sequence

Most common methods

TaqMan or intercalating dye (e.g. SYBR green)

The 5’ Nuclease Assy (TaqMan)

Two fluorescent dyes that emit at different wavelengths

target region of interest between two PCR primers

the reported (R) dye is attached at the 5’ end end of the probe sequence while the quencher (Q) dye is synthesized on the 3’ end

when the probe is intact and the reporter dye is in close proximity to the quencher dye little to no fluorescence will result

When the reporter dye molecule is released from the probe and is no longer in close proximity to the quencher dye it can begin to fluoresce

an increase in fluorescent signal results if the target sequence is complementary to the TaqMan probe

Phases of the PCR Amplification

Lag - doubling but not detected

exponential - doubling

linear - less than doubling (efficiency is dropping < 100%)

plateau - little change (efficiency is dropping < 100%)

Exponential (Geometric) Phase

during exponential amplification there is a high degree of precision surrounding the production of new PCR products

when the reaction is performing at close to 100% efficiency then there is a doubling of amplicons with each cycle

Linear Phase

since components such as deoxynucleotide triphosphates (dNTPs) or primers may be used up at slightly different rates between reactions the linear phase is not as precise from sample to sample and therefore not as useful for comparison purposes

Plateau Region

levels out

Analysis Process

The optimal place to measure is in the exponential phase of PCR

termed the cycle threshold (CT) for calculations

the fewer the cycle it gets to take to get a detectable level of fluorescence (cross the threshold), the greater the initial number of DNA molecules put into the PCR reaction

this rise in fluorescence can be correlated to the initial DNA template amounts when compared with samples of known DNA concentration (standards)

Why Real Time qPCR (advantages)

the availability of commercial qPCR kits (labs have almost entirely switched to this method of DNA quantitation))

higher throughput and reduced user interventions

qPCR will be sensitive to the same inhibitors as faced in a traditional STR test (both PCR based)

higher sensitivity

larger dynamic range

assays are target specific and can be multiplexed to a degree

Why Real Time qPCR (challenges)

qPCR is subjected to inhibition

qPCR quantitation precisions suffers at low copy numbers

when working below 100 pg qPCR is still subject to variability and uncertainty