Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien...

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Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology

Transcript of Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien...

Page 1: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Quantitative PCR Session 1: Introduction to Quantitation Methods

Presented by:

Robert O'BrienTraining Specialist – Forensic Biology

Page 2: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Quantitative PCR

Four sessions:

1. Introduction to Quantitation Methods

2. Overview of qPCR

3. Instrumentation and Software

4. Plate Preparation and Data Analysis

Page 3: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Quantitative PCR

Session 1: Introduction to Quantitation Methods

• Why quantitate DNA?• Why use Human Specific assays?• Benefits of using both autosomal and Y specific

assays• PCR nomenclature• Other quantitation methods• History of qPCR

Page 4: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?

• Quantitation shows how much DNA is available for amplification

– DNA from extraction may have to be concentrated or diluted before amplification

• Quantitation assists in troubleshooting during the analysis process

Page 5: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?

Commercial amplification kits are designed to give best results within a narrow DNA quantity range (0.5 to 2.0ng).

• Kit components are optimized for a certain amount of input DNA

• Too much or too little input DNA will adversely affect data interpretation

Page 6: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?

Common problems associated with too much input DNA:

–High stutter–Pull up–Split peaks (-A)

Page 7: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?

High Stutter

– Caused by offscale data

– Offscale data occurs when the quantity of DNA present is more than the highest level that the CCD camera of the instrument can detect

Page 8: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?Stutter is a a small peak 4 basepairs less than main peak a known artifact seen in data

The peak height of stutter is approximately 15% of main peak

Software knows the percentage of stutter peak

and determines if the small peak is a stutter peak. If it is a stutter peak it is filtered out and not assigned an allele call.

Page 9: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?• If data is offscale, the true

height of the main peak is not known

• Software cannot accurately calculate the stutter peak’s percentage of the main peak

• Therefore stutter peak is labeled as a true peak

Page 10: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?

For example if stutter at the locus D3S1358 is 10%.

Then any peak 4 basepairs before the main peak with a height of 10% or less than main peak is determinded to be stutter and is not assigned an allele call.

Page 11: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?• For example a peak is really 10,000 rfus high but

the highest peak height the software can detect is 8000 rfus. The stutter peak is 10% of true peak height, so stutter is at 1000 rfus.

• The software looks at stutter peak and determines that it is more than 10% of main peak (since it thinks main peak is only 8000 rfus). As a result it assigns the stutter peak an allele call.

In mixture interpretations this can cause problems when sorting out contributors.

Page 12: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?• Pull- up

Pull-up is when a peak of one color is seen under a peak of another color

The dyes used in kits have wavelengths of fluorescence that overlap known as spectral overlap

The software is able to correct for this spectral overlap between the dyes.

Page 13: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?• Calculation of expected

spectral overlap is based on regular peak heights.

• Spectral overlap that is not corrected for is visible in analyzed data and is called pull up

Page 14: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?

• With offscale data, spectral overlap is greater than the values calculated by the software.

• Therefore, the software subtracts out the expected amount of spectral overlap but some of it remains and it is seen as a small peak under a main peak and assigned an allele call

Page 15: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?• Pull- up from offscale data

Page 16: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?

Minus A (-A)

• Reagents in PCR have a tendency to add a 3’ nucleotide tail at the end of amplified DNA (Adenine nucleotide)

• Hold time at the end of PCR is to ensure all of the amplified DNA is given time for the addition of the poly A 3’ tail by the reagents. This ensures all peak heights are uniform. All peaks are now considered to be +A (addition of Adenine tail).

Page 17: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?

If there is too much input DNA then there is not enough time for the addition of the Adenine nucleotide to all of the amplified DNA

This results in a peak showing up in data that is one base pair shorter than the main peak

Page 18: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?• DNA without Adenine

nucleotide tail is one base pair shorter than main peak

• This appears as either– a shoulder peak (as

shown)– two peaks– a split peak

Page 19: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?• -A can also look like 2 separate peaks that are 1bp apart

Page 20: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?–A can appear as a split peak

Page 21: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?

Common problems associated with too little DNA• No data• Low level data• Lack of amplification of some loci• Locus imbalance• Allelic dropout

Page 22: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?

No data–Not enough DNA is input into PCR reaction

producing a negative result

Low level data–Peaks are seen but not enough DNA was

present for the peaks to cross the minimum threshold and be assigned an allele call

Page 23: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?Lack of amplification of some loci

– With small amounts of DNA, smaller loci are amplified and larger loci may not be amplified

– May result in a partial profile

– May erroneously appear to be degraded DNA

Page 24: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?Locus imbalance– Unbalanced amplification of two alleles at a

locus– This is caused when there is too little input

DNA and there are stochastic fluctuations in the ratio of the two different alleles

Allelic dropout

When an entire allele is missing from a profile, heterozygote may show up as a homozygote.

Page 25: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?

Page 26: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?

• Problems caused by too little or too much DNA are not realized until data interpretation:

• Causes a waste of time and resources to repeat process

• If an evidence sample was consumed in first attempt then the process cannot be repeated.

Page 27: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?

Other uses of quantitation– May determine if other amplification methods

are appropriate for testing e.g. LCN (low copy number) or mini STR testing if DNA quantity is too small.

– Can be used to troubleshoot data. – Can be used to check optimal amount of DNA

for new assays– Some quantitation methods can target specific

types of DNA (Y or mitochondrial testing)

Page 28: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?Troubleshooting with quantitation

A problem with DNA analysis is generally not seen until the profile is developed in the last stage of the DNA process.

It must be determined at what point the problem occurred in the analysis process. Problems can occur at several points and troubleshooting the entire process is very time consuming.

Page 29: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why Quantitate DNA?

An accurate and reliable means of quantitation will reduce time for troubleshooting indicating that all processes performed before quantitation (screening and extraction) were done properly.

Page 30: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Why use Human Specific assays

• Crime scenes are not clean pristine areas• DNA from non-human sources are usually

present (eg. Bacterial, animal etc.)• Human specific assays will only target human

DNA so other DNA does not affect the results• DNA audit document requires laboratories to

determine quantity of Human DNA• DAB Standard 9.3

Page 31: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Benefits of using both autosomal and Y specific assays

• Beneficial in most crimes involving a male and a female

• Evidence samples taken directly from the body of a female will typically have a lot of female cells

• If the female DNA is in a much greater quantity than male, then during total human amplification there may be little or no amplification of male DNA

• This can lead to a false negative result

Page 32: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Benefits of using both autosomal and Y specific assays

• When amplifying total human DNA. If there is too much of one person’s DNA then it is possible for that person’s DNA to mask the presence of the DNA from the other individual.

• This is commonly seen with vaginal swabs where the female DNA will overpower the male DNA if there are only a few cells from the male present.

• May result in a single source profile or the profile of the other person may be too low to be of any use.

Page 33: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Benefits of using both autosomal and Y specific assays

• Y specific assays can target only male DNA regardless of how much female DNA is present.

• This is beneficial in the following scenarios

– Sexual assault where semen is present but little or no sperm cells are present especially on vaginal swabs.

– Mixture of male and female blood at the scene or on the female where the female bled a lot more than the male

Page 34: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Benefits of using both autosomal and Y specific assays

Transfer of saliva from a male onto a female.

When swabbing the skin of a female it is possible to remove a lot of female epithelial cells in the process.

Fingernail scrapings

In these cases amplification with a Y- STR kit may be more beneficial to the case depending on ratio of quantities of total human to Y DNA

Page 35: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Benefits of using both autosomal and Y specific assays

• Y quantitation is usually done along with Total Human quantitation.

• Because when deciding how much DNA is needed for amplification the quantity of total human DNA is used.

• If the ratio of human to male is too high then conventional amplification may not be sufficient to bring up male profile.

Page 36: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Benefits of using both autosomal and Y specific assays

• Y- Quantifiler as a screening tool– High number of sexual assault cases.– The screening process is lengthy. There is

presumptive testing followed by confirmatory tests.– Some laboratories do a quick extraction of a small

amount of evidence samples and then quantitate them with total human and Y- kits.

– If male DNA is present then the evidence is put through screening process.

– If there is no male DNA then no more work on that piece of evidence is needed.

Page 37: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

PCR nomenclature

– qPCR- quantitative PCR (usually implies using PCR for DNA quantitation in “real time” not end point

– RT-PCR- Real time PCR, but in other fields often reverse transcriptase PCR (and often in conjunction with real-time PCR as well)

– Amplicon- product of PCR– Calibrant DNA- DNA of a known concentration

that is serially diluted to prepare a standard curve (can be called Standard DNA)

Page 38: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

PCR nomenclature

– Baseline- a linear function subtracted from the data to eliminate background signal

– Threshold- a value selected when the PCR is in the exponential phase of growth

– CT – Cycle threshold- The cycle number at which the amplification curve crosses the selected threshold value

– E- Efficiency- measure relating to the rate of PCR amplification

Page 39: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Other quantitation methods

• Older quantitation methods

– UV (260nm, 1 OD- 50ng/ml)– Yield gel– AluQuant– Quantiblot– Pico Green (fluorescence)– SYBR Green (Real time PCR quantitation)

Page 40: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Other quantitation methods• UV

– Uses a spectrophotometer equipped with a UV lamp

– Absorbance readings are performed at 260nm where DNA absorbs light most strongly

– From the number generated the amount of DNA present can be estimated

– 1 OD (optical density –absorbance value) is equal to 50.0 ng/ml of ds DNA

Page 41: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Other quantitation methods

• Problems with UV method

– Not very sensitive– Consumes a lot of forensic specimen– Absorbance measurements are not specific

for human DNA, it can also be influenced by contaminating proteins

– Phenol left over from the extraction procedure can give falsely high signals

Page 42: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Other quantitation methods

• Yield Gel

– DNA is loaded onto a gel– The Gel is stained with ethidium bromide

(EtBr)– EtBr intercalates in the DNA molecule and

fluoresces under UV light– Fluorescence of the sample is compared to

fluorescence given off from DNA samples of known concentrations

Page 43: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Other quantitation methods

• Problems with Yield Gel– Not sensitive– Consumes a lot of sample– Not specific to human DNA

Page 44: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Other quantitation methods

• Aluquant– Assay probes Alu repeats that are in high

abundance in the human genome– Probe target hybridization initiates a series of

enzymatic reactions that end in the production of ATP and the oxidation of luciferin resulting in the production of light

– Light intensity is read by a luminometer and is proportional to amount of DNA

– Sample quantities are determined by comparison to a standard curve

Page 45: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Other quantitation methods

• Problems with Aluquant– Cannot detect presence of inhibitors– Did not give accurate results for degraded

samples

Page 46: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Other quantitation methods

• Quantiblot- Slot blot procedure• Uses a probe that is complementary to a

primate specific alpha satellite DNA sequence D17Z1 located on chromosome 17

• Uses chemiluminescent or colorimetric detection with TMB.

• DNA is immobilized to a nylon membrane• Complementary sequences bind the

biotinylated probe• On the probe is streptavidin-horseradish

peroxidase conjugate

Page 47: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Other quantitation methods

• Streptavidin horseradish peroxidase conjugate oxidizes luminol based reagent (chemiluminescent) that emits photons or tetramethyl benzidine to produce a blue color (colorimetric)

• Quantity of DNA is determined by visual comparison between samples and a series of standards run along with samples

Page 48: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Other quantitation methods

• Problems with Quantiblot

– Results were subjective (visually comparing intensity of color from sample to standards.) People may vary on their interpretation of results. In some cases a reader was used to reduce the subjectivity of the testing.

– Time consuming and labor intensive process.

– Not able to detect PCR inhibitors

Page 49: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Other quantitation methods• Pico Green fluorescence

• Pico green is a fluorescent intercalating dye whose fluorescence is greatly enhanced when bound to double stranded DNA

• Quantitation is done by comparison to a standard curve which was constructed from standards

• Problems – Not specific for human DNA

Page 50: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Other quantitation methods

• SYBR green RT- PCR quantitation

• SYBR green system works by the detection of fluorescence given off by SYBR green dye as it binds with double stranded DNA.

• The method uses human specific primers for Alu sequences

• The more double stranded DNA that builds up from PCR the more fluorescence that is given off.

Page 51: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

Other quantitation methods

• Only problem with SYBR green system is that it binds to double stranded DNA and some extraction methods leave DNA in a single stranded form.

Page 52: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

History of qPCR

– qPCR recently developed technique– Developed by Higuchi in 1993– Used a modified thermal cycler with a UV

detector and a CCD camera– Ethidium Bromide was used as an

intercalating reporter– As concentration of double stranded DNA

increased fluorescence increased

Page 53: Quantitative PCR Session 1: Introduction to Quantitation Methods Presented by: Robert O'Brien Training Specialist – Forensic Biology.

History of qPCR

• First paper on qPCR– Higuchi, R.; Fockler,C.; Watson, R. “Kinetic

PCR analysis: real-time monitoring of DNA amplification reactions” Biotechnology (NY). 1993 Sep; 11 (9): 1026-30