# Presentation: 260416

## Training on STR Typing Using Commercial Kits and ABI 310/3100

- Margaret C. Kline, Janette W. Redman, John M. Butler
*National Institute of Standards and Technology*
- October 22-26, 2001

## Human Identity Testing

- Forensic cases -- **matching suspect with evidence**
- Paternity testing -- **identifying father**
- Historical investigations
- Missing persons investigations
- Mass disasters -- **putting pieces back together**
- Military DNA “dog tag”
- Convicted felon DNA databases

## Overview of Steps Involved in DNA Typing

## Steps in Sample Processing

- DNA
- Extraction

- DNA
- Quantitation

- PCR Amplification
- of Multiple STR markers

- _Biology_

- Separation and Detection of PCR Products
- (STR Alleles)

- _Technology_

- Sample Genotype Determination

- _Genetics_

- Comparison of Sample Genotype to Other Sample Results

- If match occurs, comparison of DNA profile to population databases

- Generation of Case Report with Probability of Random Match

**Steps in Sample Processing**

## Calculation of DNA Quantities in Genomic DNA

- _Important values for calculations_:
- 1 bp = 618 g/mol	A: 313 g/mol; T: 304 g/mol; A-T base pairs = 617 g/mol
- 			G: 329 g/mol; C: 289 g/mol; G-C base pairs = 618 g/mol
- 1 genome copy = ~3 x 109 bp = 23 chromosomes (one member of each pair)
- 1 mole = 6.02 x 1023 molecules
- Standard DNA typing protocols with PCR amplification of STR markers typically ask
- for 1 ng of DNA template.  **How many actual copies of each STR locus exist in 1 ng?**
- 1 genome copy = (~3 x 109 bp) x (618 g/mol/bp) = 1.85 x 1012 g/mol
- 		 = (1.85 x 1012 g/mol) x (1 mole/6.02 x 1023 molecules)
- 		 = 3.08 x 10-12 g = **3.08 picograms (pg)**
- Since a diploid human cell contains two copies of each chromosome, then
***each diploid human cell contains ~6 pg genomic DNA***
**** 1 ng genomic DNA (1000 pg) = ~333 copies of each locus **(2 per 167 diploid genomes)**

## PCR Process

## Thermal Cycling Temperatures

- 94 oC

- 60 oC

- 72 oC

- Time

- Temperature

*Typically 25-35 cycles performed during PCR*

**94 ****o****C**

- 94 oC

- 94 oC

- 60 oC

**60 ****o****C**

- 72 oC

**72 ****o****C**

## Thermal Cycling Parameters

## Advantages of PCR

- Minute amounts of DNA template may be used from as little as a single cell.
- DNA degraded to fragments only a few hundred base pairs in length can serve as effective templates for amplification.
- Large numbers of copies of specific DNA sequences can be amplified simultaneously with multiplex PCR reactions.
- Contaminant DNA, such as fungal and bacterial sources, will not amplify because human-specific primers are used.
- Commercial kits are now available for easy PCR reaction setup and amplification.

## Multiplex PCR (Parallel Sample Processing)

**Compatible primers are the key to successful multiplex PCR**
**10 or more STR loci can be simultaneously amplified**
**STR kits are commercially available**

- _Advantages of Multiplex PCR_
- Increases information obtained per unit time (increases power of discrimination)
- Reduces labor to obtain results
- Reduces template required (smaller sample consumed)

_**Challenges to Multiplexing**_
- primer design to find compatible primers (no program exists)
- reaction optimization is highly empirical often taking months

## Potential Pitfalls of PCR

- The target DNA template may not amplify due to the presence of PCR inhibitors in the extracted DNA
- Amplification may fail due to sequence changes in the primer binding region of the genomic DNA template
- Contamination from other human DNA sources besides the forensic evidence at hand or previously amplified DNA samples is possible without careful laboratory technique and validated protocols

## Tips for Avoiding Contamination

- Pre- and post-PCR sample processing areas should be physically separated.
- Equipment, such as pipettors, and reagents for setting up PCR should be kept separate from other lab supplies, especially those used for analysis of PCR products.
- Disposable gloves should be worn and changed frequently.
- Reactions may also be set up in a laminar flow hood, if available.
- Aerosol-resistant pipet tips should be used and changed on every new sample to prevent cross-contamination during liquid transfers.
- Reagents should be carefully prepared to avoid the presence of any contaminating DNA or nucleases.
- Ultraviolet irradiation of laboratory PCR set-up space when the area is not in use and cleaning workspaces and instruments with isopropanol and/or 10% bleach solutions help to insure that extraneous DNA molecules are destroyed prior to DNA extraction or PCR set-up

## Short Tandem Repeats (STRs)

***the repeat region is variable between samples while the flanking regions where PCR primers bind are constant***

- 7 repeats

- 8 repeats

- Homozygote = both alleles are the same length
- Heterozygote = alleles differ and can be resolved from one another

***Primer positions define PCR product size***

## STR Repeat Nomenclature

- International Society of Forensic Haemogenetics (ISFH) -- *Int. J. Legal Med.* (1997) 110:175-176
- For sequences within genes, use the coding strand
- For other sequences, select the first GenBank database entry or original literature description
- Define the repeat sequence which will provide the largest number of consecutive repeats
- If two sequences are repeated, include both motifs in determining the repeat number
- Microvariants: should be designated by the number of complete repeats and the number of base pairs of the partial repeat separated by a decimal point (Int. J. Legal Med. 1994, 107:159-160) *e.g. TH01 allele 9.3*

## Why STRs are Preferred Genetic Markers

- Rapid processing is attainable
- Abundant throughout the genome
- Highly variable within various populations
- Small size range allows multiplex development
- Discrete alleles allow digital record of data
- Allelic ladders simplify interpretation
- PCR allows use of small amounts of DNA material
- Small product size compatible with degraded DNA

## STR genotyping is performed by comparison of sample data to allelic ladders

**STR genotyping is performed by comparison of sample data to allelic ladders**

## Allelic Ladder Formation

- Separate PCR products from various samples amplified with primers targeted to a particular STR locus

## Position of Forensic STR Markers on Human Chromosomes

- 13 CODIS Core STR Loci

**Position of Forensic STR Markers on Human Chromosomes**

## Information on 13 CODIS STRs

- D18S51

- 18q21.3

- AGAA

- L18333

- 13

- 7-27

- 43

- *

## Probability of a Random Match Using 13 CODIS STR Markers

**Probability of a Random Match Using 13 CODIS STR Markers**

## Commercial STR Kits

_**Kit Contents**_:
- Allelic Ladders for Genotyping
- PCR Component Mix
- Primer Mix
- Positive Control DNA Sample

**Currently 2 Suppliers: Applied Biosystems and Promega Corporation**

**Cost to User: $15-30 per DNA sample tested**

## Value of STR Kits

- _Advantages_
- Quality control of materials is in the hands of the manufacturer
- Improves consistency in results across laboratories – same allelic ladders used
- Common loci and PCR conditions used – aids DNA databasing efforts
- Simpler for the user to obtain results
- _Disadvantages_
- Contents may not be completely known to the user (e.g., primer sequences)
- Higher cost to obtain results

**FSS: 5X higher cost with SGM Plus kit**

## Commercially Available STR Kits

- *Caucasian population (rounded to 2 significant figures)

## Same DNA Sample Run with Each of the ABI STR Kits

**Same DNA Sample Run with Each of the ABI STR Kits**

## AmpFlSTR® IdentifilerTM

**6FAM (blue)**

**VIC (green)**

**NED (yellow)**

**PET (red)**

**LIZ (orange)**

- AmpFlSTR® IdentifilerTM

## Requirements for Accurate STR Typing

- High precision (to permit comparison of allelic ladders to sequentially processed STR samples)
- Color separation of different dye sets used (to avoid bleed through between different colors)
- Resolution of at least 1 bp to >300 bp (to detect microvariants)
- Reliable sizing over 75-450 bp region

**Accurate typing can be achieved with ABI 310**

## Components of ABI 310

- Chemistry
- STR kits, fluorescent dyes, matrix samples, capillary, buffers, polymer, formamide
- Hardware
- CCD camera, laser, electrodes, pump block, hot plate for temperature control, autosampler
- Software
- Data collection, color separation, peak sizing & calling, genotyping, stutter removal

## ABI Prism 310 Genetic Analyzer

## Principles of Sample Separation and Detection

**Capillary or Gel Lane**

**Labeled DNA fragments (PCR products)**

## Chemistry Involved

- Injection
- electrokinetic injection process
- importance of sample preparation (formamide)
- Separation
- capillary
- POP-4 polymer
- buffer
- Detection
- fluorescent dyes with excitation and emission traits
- virtual filters (hardware/software issues)

## Electrokinetic Injection Process

**Electrode**

**Capillary**

## Comments on Sample Preparation

- Use high quality formamide (<100 S/cm)!
- ABI sells Hi-Di formamide
- regular formamide can be made more pure with ion exchange resin
- Deionized water vs. formamide
- Biega and Duceman (1999) *J. Forensic Sci.* 44: 1029-1031
- water works fine but samples are not stable as long as with formamide
- Denaturation with heating and snap cooling
- use 480 cycler for heating (holds 0.5 mL tubes) and cold aluminum block for snap cooling (instead of ice)
- heat/cool denaturation step is not always necessary...

## Separation Issues

- Run temperature -- 60 oC helps reduce secondary structure on DNA and improves precision
- Electrophoresis buffer -- urea in running buffer helps keep DNA strands denatured
- Capillary wall coating -- dynamic coating with polymer
- Polymer solution -- POP-4

## DNA Separation Mechanism

- Size based separation due to interaction of DNA molecules with entangled polymer strands
- Polymers are not cross-linked (as in slab gels)
- “Gel” is not attached to the capillary wall
- Pumpable -- can be replaced after each run
- Polymer length and concentration determine the separation characteristics

## Detection Issues

- Fluorescent dyes
- spectral emission overlap
- relative levels on primers used to label PCR products
- dye “blobs” (free dye)
- Virtual filters
- hardware (CCD camera)
- software (color matrix)

**Filters determine which wavelengths of light are collected onto the CCD camera**

## Laser Used in ABI 310

- Argon Ion Laser
- 488 nm and 514.5 nm for excitation of dyes
- 10 mW power
- Lifetime ~5,000 hours (1 year of full-time use)
- Cost to replace ~$5,500
- Leads to highest degree of variability between instruments and is most replaced part
- Color separation matrix is specific to laser used on the instrument

## Fluorescent Labeling of PCR Products

- Dyes are attached to one primer in a pair used to amplify a STR marker
- Dyes are coupled to oligonucleotides (primers) through NHS-esters and amine linkages on the 5’end of the primer usually through a 6-carbon spacer --- Dye-(CH2)6-primer
- Dye-labeled oligonucleotide is incorporated into PCR product during multiplex PCR amplification giving a specific color “tag” to each PCR product
- Dyes can be spectrally distinguished using virtual filters and CCD imaging to yield different colored peaks in ABI 310 electropherogram

## Dye Blobs

**DYS392**

**DYS438**

**HEX**

**DYS392**

**DYS438**

**DYS437**

*PCR product size (bp)*

- Free dye (not coupled to primer) can be injected into the CE capillary and interfere with detection of true STR alleles
- Dye blobs are wider and usually of less intensity than true STR alleles (amount depends on the purity of the primers used)
- Dye blobs usually appear at an apparent size that is unique for each dye (e.g., HEX ~170 bp)

## Fluorescent Dyes Used in 4-Color Detection

**FAM (Blue)**

**JOE (Green)**

**TAMRA (Yellow)**

**ROX (Red)**

**NED**

**FL**

**CXR**

## Fluorescent Emission Spectra for ABI Dyes

**520**

**540**

**560**

**580**

**600**

**620**

**640**

**WAVELENGTH (nm)**

**100**

**80**

**60**

**40**

**20**

**0**

**5-FAM**

**JOE**

**NED**

**ROX**

- Laser excitation
- (488, 514.5 nm)

**Normalized Fluorescent Intensity**

## Matrix File Table from an ABI 310

**Matrix File Table from an ABI 310**

## Same Dye Set and Filter F with Different ABI 310s

**Instrument lasers make a big difference**

## Primary Filter Sets on the ABI 310

**Filter A**

## If Wrong Filter Is Used...

*Y STR 10plex labeled with FAM, TET, HEX dyes*

**Filter A**

**Filter F**

**Filter C**