1. DNA Fingerprinting: RFLP & PCR
Abira Khan

2. Recap DNA profiling: RFLP DNA profiling: PCR
Steps in PCR based DNA profiling

3. Steps in STR typing by PCR
DNA Extraction
DNA Quantitation
PCR amplification of multiple STR loci (10 – 15)
Separation of PCR product by capillary electrophoresis
Data collection
Peak identification
Colour separation
Peak sizing
Comparison with allelic ladder
Genotype assignment
DNA profile

4. DNA Sample Collection
Must be collected, isolated and preserved under stringent conditions
Double swab technique for crime scene DNA collection
Buccal swab/blood collection for reference DNA
Blood, epithelial cells- Easy
Sperm- differential extraction
Hair shaft- DE, salting out, OE, PK+PCR, NaOH+filter centrifugation
Muscle tissue- Chelex, OE
Bone osteocytes, Teeth odontoblast- Cleaning, powder form by drilling/grinding under liquid nitrogen, decalcification using 0.5 M EDTA, cell lysis. DNA extraction using OE/silica. The process of extracting DNA from bone samples takes much longer than with any other type of sample
The National Institute of Justice brochure ‘What Every Law Enforcement Officer Should Know About DNA Evidence’ ( )

5. DNA Sample Storage & Characterization
Storage at 4º/-20º C, for long term at -70º C
Preliminary & confirmative test
Blood- Luminol, KM+LMG (haem)
Saliva- α amylase, mRNA profiling
Semen- Acid phosphatase (AP), or prostate specific antigen (PSA)

6. DNA Extraction Organic Extraction Chelex extraction FTA Card
Solid phase extraction- Qiagen columns, DNA IQ, PrepFiler
Differential extraction

8. DNA Quantitation A260 UV absorbance, yield gels Slot blot PicoGreen
QuantiBlot method
AluQuant
End-point PCR
Real time PCR
5’ nuclease assay
Check DNA Box 6.2

10. Importance of DNA Quantitation (prior to multiplex PCR)
DNA amount
(log scale)
High levels of DNA create interpretation challenges (More artifacts to review)
100 ng -A
Too much DNA
Off-scale peaks
Split peaks (+/-A)
Locus-to-locus imbalance +A
10 ng
2.0 ng
Well-balanced STR multiplex
1 ng
STR Kits Work Best in This Range
0.5 ng
0.1 ng
Too little DNA
Heterozygote peak imbalance
Allele drop-out
Locus-to-locus imbalance
0.01 ng
Stochastic effect when amplifying low levels of DNA produces allele dropout

11. PCR amplification of Multiple STR loci
Over Markers are Copied simultaneously
Sensitivities to levels of 1-2 ng of DNA
Different Fluorescent Dyes Used to Distinguish STR Alleles with Overlapping Size Ranges

12. PCR Components AmpliTaq Gold PCR Master Mix -10 µL
AmpliTaq Gold DNA polymerase (0.05 U/µL)
GeneAmp Gold PCR buffer (30 mM Tris/HCl, 100 mM KCl)
dNTP 400 M each
MgCl2, 5 mM
Primers set (All 16 set primers) -5 µL
Template DNA µL
Test sample: A portion of the extracted DNA is diluted in TE buffer so that 1.0 ng of total DNA is present in a final volume of 10 µL
Positive control: DNA sample with know DNA profile (supplied with the kit)
Negative control: TE buffer (10 mM Tris, 0.1 mM EDTA pH 8.0)

13. AmpliTaq Gold DNA Polymerase
AmpliTaq Gold is a thermostable DNA ploymerase to make advanced PCR techniques easy
Provided in a inactive state
Heat activates the enzyme
Allows flexibility in the PCR reaction set up, including pre-mixing of PCR reagent s at room temperature
Improves amplification of most of the templates by lowering non-specific backgrounds
Mis-primed primers will not be amplified
Activation temperature is usually well above the annealing temperature

14. Primer sets
All the primers are labeled with fluorescent dyes
The reverse primer is usually labeled
Fluorescent dyes used invariably:
FAM
JOE
NED
VIC
PET
ROX
LIZ
Most of the manufacturers do not disclose their primer sequences (except Promega Corporation)

15. Fluorescent Dyes Used in 4-Color Detection
JOE (Green)
FAM (Blue) FL
ROX (Red)
CXR
NED
TAMRA (Yellow)

16. PCR Protocol

17. COMMERCIAL THERMAL CYCLER

18. DNA Separation occurs in minutes...
Separation of PCR products by Capillary Electrophoresis (CE)
Fill with Polymer Solution
Argon Ion Laser
m x 27 cm
5-20 kV - +
Burn capillary window
Inlet (Cathode)
Outlet (Anode)
DNA Separation occurs in minutes...
Data Acquisition and Analysis

19. Principles of Sample Detection
Labeled PCR products
Sample Detection
Size Separation
CCD Panel
Detection region
Color
Separation
Laser

20. Sample preparation
Prepare a mixture of Hi-Di Formamide and size standard (LIZ) in the following ratio
Hi-Di Formamide L
Size standard 0.3 µL
Mix
1.0 µL PCR product or allelic ladder
9.0 µL formamide-size standard
Heat in thermal cycler for 3 minutes at 95ºC
Place on ice for 3 minutes

21. - - Sample injection Electokinetic injection
Capillary
Electrode
Electokinetic injection
A voltage spike is applied to pull the DNA molecules into the capillary
3100 = 3 kV/3 sec
3130 = 3 kV/10 sec
Sample Tube
DNA- -
DNA- -

22. Capillary electrophoresis instrument platforms

23. Inside view: ABI Prism 3100 Genetic Analyzer

24. ABI 3100 Array Detection

25. Capillary array

26. Laser Used in ABI 3100 Argon Ion Laser
488 nm and nm for excitation of dyes
10 mW power
Lifetime ~5,000 hours (1 year of full-time use)
Cost to replace ~$5,500

27. Sample Interpretation
Mixture of dye-labeled PCR products from multiplex PCR reaction
Sample Separation
Sample Detection
CCD Panel (with virtual filters)
Argon ion LASER (488 nm)
Color
Separation
Fluorescence
ABI Prism spectrograph
Capillary
Sample Injection
Size
Processing with GeneScan/Genotyper software
Sample Interpretation
Figure 13.8 Schematic illustration of the separation and detection of STR alleles with an ABI Prism 310 Genetic Analyzer.

28. Steps in STR Genotyping Process: computers
Data Collection
Color Separation
Data Review by Analyst/Examiner
Peak Identification
Peak Sizing
Comparison to Allelic Ladder
Confirmation of Results by Second Analyst/Examiner
Genotype Assignment to Alleles
Data Collection
software
Matrix file (Spectral calibration)
User-defined thresholds
GeneScan
software
Internal sizing standard
(e.g., GS500-ROX)
GeneMapper ID
Allelic ladder sample
Genotyper
software
Peak Editing to
Remove Artifact Calls
Expert Systems under Development (e.g., True Allele)

29. Data Collection Done by data collection software
Also controls 3100 run conditions
Translates light on CCD camera into raw data

30. Raw Data from the ABI Prism 3100
(prior to separation of fluorescent dye colors)

31. Peak identification & color separation
Peak identification (Based on threshold values)
Color separation
Sizing peaks with internal size standard

33. Overlapping markers Mobility modifying non-nucleotide linkers
To permit continued use of the same PCR primers for amplifying STR loci and still have optimal interlocus spacing within the various color channels
Changing primer sequence works too
Different primer positions have the potential to lead to allele dropout if a primer binding site mutation impacts one of the primer pairs- Concordance studies carried out

34. Genotype assignment
Comparison with allelic ladder
Genotype assignment

36. Example DNA profile

37. Example of A DNA Profile
Locus
DNA profile
Genotype Frequency (GF)
D3S1358
16, 17
2 x x = 0.125
TH01
7, 8
2 x x = 0.051
D21S11
29, 34.2
2 x x = 0.008
D18S51
12, 14
2 x 0.09 x = 0.01
Penta E
5, 15
2 x x = 0.007
D5S818
12, 12
(0.097) = 0.009
D13S317
10, 12
2 x x = 0.043
D7S820
(0.229) = 0.052
D16S539
11, 12
2 x 0.32 x = 0.135
CSF1PO
10, 11
2 x x = 0.102
Penta D
9, 12
2 x x = 0.135
vWA
18, 18
(0.195) = 0.038
D8S1179
13, 15
2 x x =
TPOX
8, 8
(0.358) = 0.128
FGA
20, 25
2 x x = 0.042
Amelogenin
X, Y
Probability of Match = 1.3 x ( 1 in Sextillion)

38. PCR Based DNA Typing Advantages of PCR based method:
Very small amounts of DNA template may be used, even as little as from a single cell
DNA degraded to fragments only a few hundred base pairs in length can serve as an effective template for amplification
Large numbers of copies of specific DNA sequences can be amplified simultaneously with multiplex PCR reactions
Contaminant DNA, such as from 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

39. PCR Based DNA Typing Disadvantages of PCR based method:
The target DNA template may not amplify due to the presence of PCR inhibitors in the extracted DNA
Amplification may fail due to sequence mutations in the primer binding region of the genomic DNA template- something often referred to as a ‘Null allele’
Contamination from other human DNA sources besides the forensic evidence at hand or previously amplified DNA samples is possible

40. Challenges in Forensic Science
Degraded DNA- miniSTR
Mixture
Chromosomal abnormalities
LCN- Increased PCR cycles
Contaminations
PCR inhibitors
Abnormal peaks in DNA profile

41. PCR Inhibition
Interferes with the cell lysis necessary for DNA extraction
Interferes by nucleic acid degradation or capture
Inhibits polymerase activity, thus preventing enzymatic amplification of the target DNA
Bovine serum albumin (BSA) prevents/reduces PCR inhibition

42. Extra Peaks
Biological Artifacts: Stutter products, Split peak/Incomplete 3’ (+A) nucleotide addition, Triallelic patterns, Mixed sample results, Allele dropout, Too much/little sample DNA
Technology related Artifacts: Matrix (multicomponent) failure/Pull up, Dye blobs, Air bubbles, urea crystals , or voltage spikes, Sample contaminants, Overloaded profile (Too many PCR cycles), Peak heights
Check Chapter 7 of Goodwin

43. COMPARISON OF METHODS
Check table 3.6 from Fundamentals of forensic DNA typing- Butler

44. REFERNCE Fundamentals of Forensic DNA Typing- John M. Butler
Forensic DNA Typing, Biology, Technology, and Genetics of STR Markers- John M. Butler- 2nd edition
An Introduction to Forensic Genetics- William Goodwin