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

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

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

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

Why Quantitate DNA? Common problems associated with too much input DNA: –High stutter –Pull up –Split peaks (-A)

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

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.

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

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.

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.

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.

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

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

Why Quantitate DNA? Pull- up from offscale data

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).

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

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

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

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

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

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

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

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.

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.

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)

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.

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.

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

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

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.

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

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

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.

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.

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)

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 –C T – 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

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)

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

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

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

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

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

Other quantitation methods Problems with Aluquant –Cannot detect presence of inhibitors –Did not give accurate results for degraded samples

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

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

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

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

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.

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.

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

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) Sep; 11 (9):