Forensic Bio DNA Quantitation

What is the purpose of DNA quantification?

To determine how much DNA has been extracted from a sample

How much DNA does standard DNA profiling require?

.5-1.0 ng

What are the 3 common quantification methods?

1. Spectrophotometry (UV absorbance)
2. fluorescence tests
3. Quantitative real time PCR (qPCR)

How does Picogreen work?

It is an ultra-sensitive fluorescent nucleic acid stain
When bound to dsDNA exhibits 1,000 fold fluorescence

Fluorescence vs. UV Absorption

Fluorescence:

10,000 fold more sensitive than UV detection range
Highly sensitive for dsDNA with little interference
No assessment of purity

UV Absorption:

Relative insensitivity of the assay detection range
Can have interference in signal level from contaminants
Purity assessment possible

Who devised PCR?

Kary Mullis

What are parts of a PCR?

Primers that flank the DNA to show what section of it needs to be copied

DNA Polymerase to read the DNA and copy it

Nucleotides to build the copy of the DNA

Buffer solution to create optimal conditions for the polymerase activity

Mg2+ ions

What are the 3 basic steps of PCR?

1. Denaturation
2. Annealing
3. Extension

Does PCR amplify DNA exponentially? True/False?

True

What direction do PCR primers direct DNA polymerase in?

5' to 3'

What is the purpose of Hot Start PCR?

to inhibit Taq DNA polymerase activity

Who developed qPCR?

Higuchi

Why is qPCR called "real time" PCR?

Since it allows detection of the amplicon as it accumulates during the PCR cycle, not only at the end like regular PCR

Is fluorescence involved in qPCR?

Yes, a fluorescent molecule is included in the PCR assay and fluorescence is measured after every PCR cycle producing amplification curves

Increase in fluorescence is proportional to the accumulation of the amplicon

What are the 3 phases of qPCR amplification?

1. Exponential
2. Linear
3. Plateau

What is Cycle Threshold (CT)?

Number of PCR cycles to reach threshold where fluorescence detected

What phase of qPCR is the CT placed in?

Exponential phase as this is where the reaction is amplifying

What is absolute quantitation?

Determining the exact DNA copy number

What is SYBR Green?

binds double stranded DNA and as
more double stranded DNA is copied with each round of qPCR there are more DNA copies to bind SYBR Green which increases amount of fluorescent light emitted

What are Taqman probes?

Reporter dye attached to 5' end; Quencher attached to 3' end

During PCR extension the probe is displaced and the reporter is cleaved off resulting in fluorescence

Taqman vs. SYBR Green

Taqman:

Highly specific
Higher sensitivity
Multiplex possible
More Complex

SYBR Green:

Not specific
Cannot multiplex
Simple design
Little optimization required

What are the 3 parts of a quantifiler trio?

Small autosomal target
Large autosomal target
Y-chromosomal target

What does the IPC do?

Internal positive control

Short strand of DNA that is amplified with your samples

Shows levels of inhibition, assesses quality of your sample

If the IPC DNA amplifies, but your sample does not?

Low amount or no starting template

If the IPC DNA and your sample don't amplify?

Indicates inhibition of the PCR

What does comparing the CT of the IPC to the CT of the NTC do?

Reduced CT of the IPC can indicate some level of inhibition

Degradation Assessment

Degradation index

Small autosomal target concentration
Large autosomal target concentration

Mixture Assessment

Indicates whether a male/female mixture is present
by comparing the ratio of autosomal DNA to Y-chromosomal DNA

Mixture ratio:

Male DNA : Female DNA
Female DNA calculated by: Total human DNA conc. - Total Male DNA conc.
Is useful in deciding whether to proceed with autosomal STR or Y-STR analysis