FS531L: Advanced Forensic Biology Lab: Pre-Lab Quiz 2

What are the steps in DNA analysis?

1) Extraction

2) Quantitation

3) Amplification

4) Separation

5) Analysis and Interpretation

Degraded DNA has...

less amplifiable DNA

What can block the PCR process?

inhibitors (internal, substrate, chemical) -- note: nowadays most chemical kits include reagents to help prevent this

How can too much DNA affect PCR amplification?

it can block the reaction

How can too little DNA affect PCR amplification?

can cause multiple issues with further analysis

i.e. allele dropout (where you see strange results due to too much reagent as compared to DNA)

What is an example of something that can lead to bad quant?

pipetting error

The type of quantitation used depends on...

- what question is being asked (what are you measuring?)

- what type of DNA testing is being performed (RFLP or PCR?)

Quantitation must be done because...

it is a requirement of the DNA Advisory Board (DAB) -- requires human-specific DNA quantitation, so that appropriate levels of human DNA can be included in the subsequent PCR amplification

What is the molecular weight of a DNA base pair?

Of each of the nitrogenous bases?

- 618 g/mol

A: 313 g/mol

T: 304 g/mol

A-T: 617 g/mol

G: 329 g/mol

C: 289 g/mol

G-C: 618 g/mol

What is the molecular weight of DNA?

1.85 x 10^12 g/mol

How much DNA does a diploid (and haploid) human cell contain?

~ 6 pg genomic DNA (diploid)

- ~3.08 pg genomic DNA (haploid)

One ng of DNA contains the DNA from how many cells?

How many copies from each locus?

~ 167 diploid cells

~ 333

The method used determines...

technology

RFLP v.s. PCR

RFLP:

- required the quant. of the total amount of DNA present

- all DNA (huma, bacterial, fungal, etc.)

PCR:

- requires that you know the amount of target species DNA present

* primers are usually genera/species specific

* normally threshold range of DNA as test validated

* originally as high as a microgram, now in ng range

What is the optimal DNA input range for most STR kits today?

0.5 - 2 ng

What can happen with too much DNA?

Too little?

Too much DNA:

- overblown peaks

- incomplete adenylation (forked peaks)

- affects on stutter bands

- locus to locus imbalance

- no or poor amp due to primer saturation

Too little DNA:

- stochastic effects

- allele dropout

- allele dropin

- heterozygote imbalance

- locus to locus imbalance

What causes stutter band formation?

slippage of DNA polymerase

Why is DNA quantitation important prior to multiplex PCR?

High levels of DNA create interpretation challenges (more artifacts to review)

Low levels of DNA produce stochastic effect when amplifying low levels of DNA -- produces allele dropout

What is all quantitation based on?

comparison to a standard curve; the unknown compound is compared to known samples

Quantitation can be ? or ? depending on analyte

linear or non-linear

Discuss how UV absorbance is used in DNA quant.

- UV Spectrophotometer 260/280 ratio and absorbance can measure DNA quantity and purity

- DNA absorbs UV at 260 nm, aromatic amino acids absorb at 280 nm.

- Ratio > 1.80 indicates relatively pure DNA

What were the issues/problems with UV absorbance for DNA quant?

- required too much sample for forensic use; also time-consuming without automation

* new nanodrop uses a drop of sample, but is not highly accurate/reliable

Problems:

- if using phenol-chloroform (organic ex) and have phenol in DNA, it will read high

- measures all DNA

- does not tell you if DNA is degraded

UV absorbance quant is best for...

pristine samples (paternity testing, known samples, research)

What wavelength does DNA absorb UV? Aromatic amino acids?

DNA: 260 nm

AAA: 280 nm

What ratio for UV absorbance indicates relatively pure DNA?

> 1.80

What is the Yield Gel and Gel-Pro Quant system? How does it work?

- NIST RFLP yield

- Gel kit (SRM 2390) to quant DNA for RFLP testing

- visual comparison of unknown bands to known bands gives you an approximation

- can scan gel, put in known DNA samples and get more accurate results

- will detect degraded samples (based on band intensity)

What is Second Generation Quantitation? How does it work?

Slot Blot

- DNA sample is denatured with NaOH and blotted onto nylon membrane

- neutralized

- Probed with 40 bp D17Z1 probe (primate specific)

- original dot/slot blots used radioactive labels, later switched to colorimetric or chemiluminscenet detection

Discuss the advantages/disadvantages, etc. of slot blots.

- Relative quant. by visual comparison (subjective)

- Scan blot and computer estimation, more accurate

- Used about 5 uL of extracted DNA

- Detected single and double stranded DNA

- Lower limit about 150 pg (30 pg/ul)

- ABI stopped selling and supporting in 2006

What do intercalating assays use?

- ethidium bromide

- Hoechst 33258

- SYBR Green I

- PicoGreen

How do intercalating assays work?

use intercalating dyes that bind to dsDNA and will fluoresce when excited by UV or specific frequency light

PicoGreen and SYBR Green bind to...

ssDNA about 10 fold less efficiently than dsDNA.

How to set up a quantitation test for intercalating assays?

1) Use 96 well black microplates

2) 5 uL of sample is diluted in 195 uL of PG or SG reagent

3) 80 samples and 16 calibrants are put on microplate

4) Sample analysis and generation of quant can be done in 30 min

5) Reported LOD range from 0.025-0.25 pg/uL

Intercalating assays always require...

fluorescent microplate readers

What are the pros and cons of intercalating assays?

Pros:

- fast

- reagents about 20 cents

- relatively biohazard free

Cons:

- cost of reader

- measures all DNA

- does not tell you about degradation

What is the AluQuant Human DNA quant System?

- probe binds to Alu sites (primate specific)

- does not require PCR

- enzymatic steps lead to ATP production that oxidizes luciferin to produce light which can be measured on a luminometer.

Standard Curve range for AluQuant?

0.1 - 50 ng

AluQuant was replaced by...

real-time quantitative PCR

What is end point PCR? How does it work?

uses amplification of a single target DNA locus, such as Alu

- uses SYBR green as marker of amplified DNA

- done in regular PCR machine (exponential, linear, plateau phase)

- detected as RFUs on regular detection (ABI 310, 3100, 3130)

- compared to std. curve generated by known DNA samples

What is significant about end-point PCR?

monitors PCR

Overall Summary of Initial Quantification Methods?

What are their drawbacks?

- UV (260/280)

- Yield Gel

- Slot Blot

- AluQuant

- Intercalating Assays (PG, SG) - fluorescence

- others

Drawbacks:

- time-consuming (multiple steps)

- not connected to software analysis

- limited dynamic range

- some not human specific

What is the latest quant method for quantitation of DNA?

Real-time PCR

What are the main goals/takeaways for DNA quantitiation?

Would like quantitation to:

- measure human DNA

- determine amplifiable DNA

Answer:

- Are inhibitors present?

- Is the DNA degraded? (Degradation index)

- Is the quantity too low to amplify?

When was qPCR invented and by whom?

originally described by Russ Huguchi in 1990s

- called quantitative PCR

How does qPCR work?

monitors cycle to cycle fluorescence in a closed tube system

- two approaches: SYBR Green measures formation of dsDNA (mostly)

What is the commonly used instrument for qPCR?

ABI 7500 Real-Time PCR

How does the ABI 7500 Real-Time PCR instrument operate?

- TaqMan System (ABI) uses displacement of dual dye labeled probe, leading to fluorescence

- Uses 5' exonuclease activity of Taq polymerase to cleave probe

What is Taqman probe?

Consists of a fluorophore covalently attached to the 5' end of the oligonucleotide probe and a quencher at the 3' end. The quencher molecule quenches the fluorescence emitted by the fluorophore when excited by the cycler's light source via FRET. As long as the fluorophore and the quencher are in proximity, quenching inhibits any fluorescence signals. TaqMan probes are designed such that they anneal with a DNA region amplified by a specific set of primers. As the Taq polymerase extends the primer and synthesizes the nascent strand, the 5' to 3' exonuclease activity of the polymerase degrades the probe that has annealed to the template. Degradation of the probe releases the fluorophore from it and breaks the close proximity to the quencher, thus relieving the quenching effect and allowing fluorescence of the fluorophore. Hence, fluorescence detected in the quantitative PCR thermal cycler is directly proportional to the fluorophore released and the amount of DNA template present in the PCR

What are the main steps of the Taqman assay?

1) Polymerization and Strand Displacement

2) Probe Cleavage (release of reporter dye)

3) Completion of Polymerization

When does fluorescence occur in the TaqMan assay?

Fluorescence occurs when reporter dye and quencher dye are no longer in close proximity

What is the function of the exonuclease in qPCR analysis?

- frees reporter dye from quencher dye

In qPCR, fluorescence is...

correlated with DNA concentration

How are qPCR results interpreted?

as a plot of fluorescence v.s. cycle number

What are the thee phases of the fluorescence v. cycle number plot?

1) exponential

2) linear

3) plateau

Where is qPCR data most accurate?

exponential phase

What is Ct?

point where fluorescence is detected/reported by the instrument

CT is in a sense...

arbitrary

How is the standard curve used in qPCR analysis?

- want to know cycle number that CT was crossed

- standard curve relates cycle number to quantity of log DNA concentration.

By knowing cycle number, you can estimate concentration.

What are some factors that affect qPCR results?

- more degraded DNA leads to lower quantity of amplifiable DNA

- more inhibitor -- greater deviation of amplification (internal) control expected value

- greater number of cycles CT, lower DNA concentration

- accuracy of diluting and pipetting standards (inter- and intra-individual)

- accuracy of standard

- accuracy of pipetting unknown

- high variation between labs reflects inherent error in the methodology

More degraded DNA =

lower quantity of amplifiable DNA

More inhibitor =

greater deviation of amplification (internal) control expected value

The greater number of cycles to CT in qPCR

LOWER DNA CONCENTRATION

What is contained in the Quantifiler Trio Kit?

- dye in kit for pipetting

- IC probe for inhibition

- nuclear probe

- Y probe

- Large fragment probe to measure degradation

- reagents, primers, IPC, probes

The Quantifiler Trio Kit uses ? points for the std curve

5 different points for the std curve (5 pg - 50 ng)

Quantifiler Trio Kit:

Target Loci:

Optimized Chemistry:

DNA Standard:

Target Loci: uses multiple-copy target loci for improved detection sensitivity. Three human-specific target loci: Small Autosomal (SA), Large Autosomal (LA) and Y-chromosome target.

Optimized Chemistry: more efficient multiplexing, faster PCR cycle times (~ 1 hour), and a better inhibitor tolerance

DNA Standard: pooled, human genomic, smaller MW DNA for improved stability, homogeneity, and correlation with forensic samples

What is the PlexorHY Quencher System?

- Powerquant 5 color run on ABI 7500

- No data on technology

- Appears to be a Taqman assay

- PlexorHY-different technology and colors can be run on ABI 7500.

* instead of using increased fluorescence

* uses a quencher system

* Iso-dCTP with 5'-fluorfor

* Dabeyl-iso-dGTP quencher

What is the Investigator Quantiplex HYres Kit?

detection of amplification is performed using Scorpions primers and a novel, fast PCR chemistry

These primers are bifunctional molecules containing a PCR primer covalently linked to a probe

Fluorophore in this probe directly interacts with a quencher -- which reduces fluorescence.

It is when the quencher and fluorophore are no longer in close proximity that we see fluorescence

What are the advantages of real-time PCR?

- Availability of commercial qPCR kits (labs are now switching over to these kites)

- higher throughput and reduced user intervention -- automated set up

- simple data analysis

- experimental data rapidly analyzed in software

- interpolating into the calibration curve

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

- no post PCR manipulation (reduced contamination issues)

- high sensitivity (down to a single copynumber)

- large dynamic range (~30 pg to 100 ng)

- assays are target specific (autosomal, mito, Y) and can be multiplexed (to a degree)

What are the challenges of real-time PCR?

- qPCR is subject to inhibition

- internal PCR controls (IPC) can help

- qPCR quant precision suffer from lowcopy numbers (below 30 pg by a factor of 2)

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

-qPCR quantitates specific target sequences-- it does not quantify DNA

- in highly degraded samples, assays that amplify short target sequences will detect and measure more DNA than assays that amplify long target sequences (relevant to STR typing)

- accurate qPCR quant assumes that each unknown sample is amplified at the same efficiency as the calibrant sample in the dilution series

- result are relative to the calibrant

Every run should have...

a minimum of two (2) replicates of each standard and samples, and NTC (non-template controls)

What does the slope indicate? What slope is a good example?

- PCR efficiency

around -3.3 means good (100% or close to) PCR efficiency

What is the R^2 value a measure of?

a measure of the closeness of fit between the standard curve regression line and the individual CT data points

What are NTCs and why must they be evaluated?

non-template controls; assessed to determine the acceptability of the assay

What is an IPC? How can it be used?

internal PCR control; synthetic DNA template that is a component of the MasterMix

The IPC can be used to indicate true negative sample results from reaction affected by the presence of PCR inhibitors, assay setup, and chemistry or instrument failure.

Expected/acceptable IPC values?

20-30

What is indicative of a true negative result?

True negative results occur when no human DNA is detected (no signal for the SA, LA, and Y targets) and the IPC CT value falls within the expected range.

What is indicative of a complete PCR amplification failure?

Undetected results for all targets (SA, LA, Y, and IPC) indicates a complete PCRa mplification failure. This result is considered invalid, and the sample should be re-quantified

What does no or weak amplification of the IPC target indicate?

No amplification or weak amplification (CT > 30) of the IPC target could indicate PCR inhibition. In addition, suppressed amplification of the SA, LA, and Y targets (CT > 30)can occur due to PCR inhibition. This is typically more pronounced in the LA target because it is more susceptible to inhibitory effects

What can larger DNA concentrations cause?

Larger DNA concentrations (> 5 ng/μL) can cause competition between the human and IPC PCR reactions resulting in inhibitory effects of the IPC target (CT > 30)

Evaluating the IPC results for the different targets can help make certain decision prior to proceeding to STR analysis.

Some decisions may include:

1. Proceed directly to STR analysis

2. Dilute the sample prior to STR analysis

3. Remove PCR inhibitors

4. Or select a more robust amplification test kit that is better suited for PCR inhibitors

What is the primary quantitation value that should be used for determining STR input?

small autosomal target (SA)

After viewing and evaluating the quality of the results, samples containing a SA or Y target value of less than...

0.002 ng/μL should not proceed directly to STR analysis

If the results indicate insufficient DNA for STR analysis, the following options are available:

1. Drop the sample from the workflow and report the results as is.

2. Drop the sample from the workflow and re-extract the sample.

3. Perform concentration on the sample

Samples having a Male: Female ratio less than...

1:20 (e.g., 1:25, 1:50, 1:100) may benefit from using Y-STR analysis rather than autosomal-STR analysis.

**Specific case information should be used when deciding if Y-STR analysis is appropriate

What can be used to determine the quality index of a sample?

IPC CT flag and degradation index

What is the degradation index and what does it tell you?

The degradation index refers to the data observed when a sample displays a decrease in measured amount for large DNA fragments compared to small DNA fragments.

- can be used as a general indicator of whether large DNA fragments may perform more poorly relative to small DNA fragments during STR analysis

When the large autosomal (LA) target is undetermined...

this can be an indication of significant degradation and/or inhibition.

What can affect the degradation index?

The degradation index can be affected by the degree of degradation of the LA target and/or the presence of PCR inhibitors

PCR inhibitors that decrease the performance of the LA target in comparison to the SA target, cause...

less efficient amplification and larger CT values for the LA target.

The degradation index should be evaluated alongside...

the IPC CT results

When the IPC CT flag is triggered, what does it usually indicate?

it typically indicates the presence of PCR inhibitors in sufficient concentration to significantly impact STR analysis

If < 1, is there likely degradation?

no, not likely whether IPCCT flag is triggered or not

If the IPCCT is not triggered and DNA degradation index is > 10, what does this mean?

typically indicates DNA is significantly degraded, can also indicate PCR inhibitors.

If IPCCT flag is triggered and degradation index is >1, what does this mean?

likely degraded and/or affected by PCR inhibitors