First 4 chapter slides. Notes on First 4 chapters

1. Forensic DNA Typing
By JOHN M.BUTLER

2. Muhammad Ahmad
Bachelor of Science in Bioinformatics
OMSATS Institute of Information Technology
COMSATS Road, off G. T Road, Sahiwal.

3. Chapter 1
Overview and History of DNA Typing
©2002 Academic Press

4. History of forensic DNA analysis
 Described by Dr.Alec Jeffrey
 There are repeated regions over and over again next to each other.
 The number of repeated regions differ from individual to individual.
 These regions are known as VNTRs.
 RFLP is used to examine VNTRs.

5. Comparison of DNA Typing Methods
 The efficiency of methods depends on
1. Ability to differentiate two individuals
2. Speed with which results are Obtained

6. Comparison of DNA Typing Techniques
Speed of Analysis
(Technology)
Power of
Discrimination
(Genetics)
Low
High
Slow Fast
Markers Used
(Biology)
RFLP
Single Locus Probes
RFLP
Multi-Locus Probes
ABO
blood groups
Multiplex STRs
DQ
single STR
D1S80
mtDNA
PolyMarker
©2002 Academic Press

7. mtDNA Importance
 As in previous shown quadrant mtDNA has lowest efficiency
 But it can be very helpful in cases where severely DNA samples are involved
or in maternally related cases.

8. Sample Obtained from
Crime Scene or Paternity
Investigation
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

9. Separation and Detection Methods
1. Separation Methods
I. Slab gel
II. Capillary Electrophoresis: used to separate ionic species by their charge
and frictional forces and hydrodynamic radius
2. Detection methods
Fluorescence detection methods are used
ABI Prism 310 Genetic Analyzer

10. Chapter 2
DNA BIOLOGY REVIEW

11. Basic Concept
 DNA is our genetic blue- print .
 DNA provides a computer program that determines our physical features and
many other attributes.
 The entire DNA in a cell, is referred to collectively as its genome.
Two primary purposes of DNA
 to make copies of itself so cells can divide and carry on the same
information.
 To carry instructions on how to make proteins so cells can build the
machinery of life.

12. Structure of DNA
 DNA is double helix.
 DNA is composed of nucleotides
 Nucleotides are made up of
 Nucleobase(A,T,G,C)
 Sugar
 Phosphate

13. Base Pairing of DNA Strands
A = T
G C
T = A
A = T
C G
T
C
C
A
G
G
T
A
G C
T = A
T = A
C
G
A = T
A = T
G
C
5’
3’
5’
3’
5’ 3’
3’ 5’
denatured
strands
hybridized
strands
Hydrogen bonds
C
G C
G
G
C
Phosphate-sugar
backbone
©2002 Academic Press
5’end
|
Phosphate
|
Sugar—Base…
|
Phosphate
|
Sugar—Base…
|
3’end

14. Target Region for PCR
chromosome
cell nucleus
Double stranded
DNA molecule
Individual
nucleotides
DNA in the Cell

15. Chromosomes
• DNA found in the nucleus of the cell (nuclear DNA) is divided into chromosomes,
which are dense packets of DNA and protection proteins called histones. The
human genome consists of 22 matched pairs of autosomal chromosomes and two
sex determining chromosomes.
• Most human identity testing is performed using markers on the autosomal
chromosomes and mitochondrial DNA while gender determination is done with
markers on the sex chromosomes.

16. Some concepts….
 Homologous: If the pair of chromosome has same size and same genetic
material.
 Heterologous: If the pair of chromosome do not have same size and genetic
material.
 Mitosis: Mitosis is the process of nuclear division in somatic cells that
produces daughter cells, which are genetically identical to each other and to
the parent cell.
 Meiosis: Meiosis is the process of cell division in sex cells or gametes. In
meiosis, two consecutive cell divisions result in four rather than two daughter
cells, each with a haploid set of chromosomes.

17. cont….
 Euchromatin: Regions of chromosomes that are transcriptionally active are
known as euchromatin.
 Heterochromatin: Regions of chromosomes that are transcriptionally inactive
are known as heterochromatin.
 Karyotype: The characterization of the chromosomal complement of an
individual or a species, including number, form, and size of the chromosomes.

18. Chromosome Structure
 Centromere: The center region of a chromosome,
controls the movement of the chromosome during cell
division.
 On either side of the centromere are „arms‟ that
terminate with telomeres. The shorter arm is referred
to as „p‟ while the longer arm is designated „q‟.
p
(short arm)
centromere
telomere
q
(long arm)
telomere
Band 5
Band 3
Chromosome 12
12p3
12q5

19. Numbers of Chromosome reasoning
 chromosomes are numbered based on their overall size with chromosome 1 being the largest and
chromosome 22 the smallest.
chromosome staining
• A common method for staining chromosomes to obtain a banding pattern is
the use of a Giemsa dye mixture that results in so-called ‘G-bands’ via the
‘G-staining’ method. These G-bands serve as signposts on the chromosome
highway to help determine where a particular DNA sequence or gene is
located compared to other DNA markers.

20. Genes
 The coding regions are known as genes and contain the information necessary
for a cell to make proteins. Humans have less than 30000 protein coding
genes.
 Two parts of a gene
o Exons: coding region of gene
o Introns: non-coding region of gene.
 Only 5% of human genome are genes while 95% “junk DNA”.

21. Some concepts about Genes…
 Locus: location of gene or marker on DNA in known as Locus.
 Alleles: alternative possibilities for a gene are known as alleles.
 Homozygous: if alleles are identical e.g. AA.
 Heterozygous: if alleles are different e.g. Aa.
 Genotype: A genotype is a characterization of the alleles present at a genetic
locus.
 DNA Profile: A DNA profile is the combination of genotypes obtained for
multiple loci. DNA typing or DNA profiling is the process of determining the
genotype present at specific locations along the DNA molecule.

22. Markers
 A marker is a gene or DNA sequence with a known location on a chromosome that can be used to
identify individuals or species.
 Markers used for human identity testing are found in the non-coding regions either between
genes or within genes (i.e., introns).
 The most recent and probably most rap- idly accepted forensic DNA markers are short tandem
repeats (STRs).

23. ADVANTAGES OF STR MARKERS
 They are PCR-based and work with low-quantity DNA templates or degraded
DNA samples.
 They are amenable to automation and involve sensitive fluorescent detection,
which enables scientists to collect data quickly from these markers.
 STRs are highly discriminating between unre- lated and even closely related
individuals.

24. Nomenclature For DNA Markers
 Two ways for naming a marker
o If a marker is part of a gene or falls within a gene, the gene name is used in
the designation. For example TH01. Sometimes the prefix HUM- is included at
the beginning of a locus name to indicate that it is from the human genome.
So HUMTH01.
Here
TH name of gene i.e human tyrosine hydroxylase.
01 The intron number of gene. i.e intron number 1 of gene.

25. Cont……
o If DNA markers fall outside of gene regions may be designated by their
chromosomal position. For example D5S818.
Here
D DNA
5 Chromosome number i.e. 5
S Single stranded
818 The number of locus on chromosome

26. Product rule..
 If each locus is inherited independent of the other loci, then a calculation of
a DNA profile frequency can be made by multiplying each individual genotype
frequency together. This is known as the product rule.

27. GENETIC VARIABILITY
 With larger numbers of alleles for a particular DNA marker, a greater number
of genotypes result. In general, if there are n alleles, there are n homozygous
genotypes and n(n − 1)/2 heterozygous ones.
 For example a locus with ten possible alleles would exhibit 10 homozygous
possibilities plus [10×(10−1)]/2 heterozygous possibilities or 10 + 45 = 55 total
genotypes.

28. Recombination
 Recombination is the process by which progeny derive a combination of genes
different from that of either parent.
 In this manner, human genetic material is effectively shuffled with each
generation producing the diversity seen in the world today.

29. GENBANK: A DATABASE OF DNA SEQUENCES
 Genetic variation from DNA sequence information around the world is
cataloged in a large computer database known as GenBank. GenBank is
maintained by the National Center for Biotechnology Information (NCBI).
 As of December 2003, GenBank contained over 36 billion nucleotide bases
from more than 30 million different records.
 This repository of DNA sequence information is not from humans alone. Over
120000 different species are represented in GenBank.

30. METHODS FOR MEASURING DNA VARIATION
 Primary approaches for performing DNA typing
 Restriction fragment length polymorphism (RFLP),
 Polymerase chain reaction (PCR)-based methods.

31. Characteristic RFLP Methods PCR Methods
Time required to obtain
results
6-8 weeks with radioactive
probes; ~1 week with
chemiluminescent probes
1-2 days
Amount of DNA needed 50-500 ng 0.1-1 ng
Condition of DNA needed high molecular weight,
intact DNA
may be highly
degraded
Capable of handling
sample mixtures
Yes (single locus
probes)
Yes
Allele identification Binning required Discrete alleles
obtained
Power of Discrimination ~1 in 1 billion with 6 loci ~1 in 1 billion with 8-
13 loci (requires
more loci)
Comparison of RFLP and PCR

32. Chapter 3
Sample Collection and Preparation
(DNA Extraction and Quantification)

33. DNA Sample Sources
 The most common materials in forensic laboratories are
 Blood
 Bloodstains
 Semen
 semen stains.

34. Blood and blood stains Budowle 1995
Semen and semen stains Budowle 1995
Bones Gill 1994
Teeth Alvarez 1996
Hair with root Higuchi 1988
Hair shaft Wilson 1995
Saliva (with nucleated cells) Sweet 1997
Urine Benecke 1996
Feces Hopwood 1996
Debris from fingernails Wiegand 1993
Muscle tissue Hochmeister 1998a
Cigarette butts Hochmeister 1991
Postage stamps Hopkins 1994
Envelope sealing flaps Word 1997
Dandruff Herber 1998
Fingerprints Van Oorschot 1997
Personal Items: Razor blade,
chewing gum, wrist watch, ear wax,
toothbrush
Tahir 1996
Material Reference
Sources of Biological Evidence
©2002 Academic Press

35. Cont…
 DNA evidence collection from a crime scene must be performed carefully and
a chain of custody established in order to produce DNA profiles that are
meaningful and legally accepted in court.
 The evidence must be carefully collected, preserved, stored, and transported
prior to any analysis conducted in a forensic DNA laboratory.
 The National Institute of Justice has produced a brochure entitled „What
Every Law Enforcement Officer Should Know About DNA Evidence‟ (see
http://www.ojp.usdoj.gov/nij) that contains helpful hints for law
enforcement personnel who are the first to arrive at a crime scene.

36. Suggestions for Sample Collection
 Avoid contaminating the area where DNA might be present by not touching it with
your bare hands, or sneezing and coughing over the evidence.
 Use clean latex gloves for collecting each item of evidence. Gloves should be changed
between handling of different items of evidence.
 Each item of evidence must be packaged separately.
 Bloodstains, semen stains, and other types of stains must be thoroughly air-dried prior
to sealing the package.
 Samples should be packaged in paper envelopes or paper bags after drying. Plastic
bags should be avoided because water condenses in them,
 Stains on unmovable surfaces (such as a table or floor) may be transferred with sterile
cotton swabs and distilled water.

37. COLLECTION OF REFERENCE DNA SAMPLES
 In order to perform comparative DNA testing with evidence collected from a
crime scene, biological samples must also be obtained from suspects or
convicted felons.
 Family reference samples are used in missing persons investigations,
paternity testing, and mass disaster victim identifications.

38. PRESUMPTIVE TESTS
 Prior to taking the effort to extract DNA from a sample, presumptive tests are
often performed to indicate whether or not biological fluids such as blood or
semen are present on an item of evidence (e.g., a pair of pants).
 Three primary stains of forensic interest come from blood , semen, and
saliva. Identification of vaginal secretions, urine, and feces can also be
important to an investigation.
 Serology is the term used to describe a broad range of laboratory tests that
utilize antigen and serum antibody reactions. For example, the ABO blood
group types are determined by serology.

39. DETECTION OF BLOOD STAINS
 Blood is composed of liquid plasma and serum with solid components
consisting of red blood cells (erythrocytes), white blood cells (leukocytes),
and platelets (thrombocytes).
 Most presumptive tests for blood focus on detecting the presence of
hemoglobin molecules, which are found in the red blood cells.
 Two test are used for human blood testing
 A simple immunochromatographic test
 Luminol test

40. Cont…
 A simple immunochromatographic test for identification of human blood is
available from Abacus Diagnostics (West Hills, CA) as the ABAcard®
HemaTrace® kit.
 This test has a limit of detection of 0.07 μg hemoglobin/mL and shows
specificity for human blood along with higher primate and ferret blood.
 Luminol test can be used to locate traces of blood that have been diluted up
to 10 million times.
 The luminol reagent is prepared by mixing 0.1 g 3-amino-phthalhydrazide and
5.0 g sodium carbonate in 100 mL of distilled water. Before use, 0.7 g of
sodium perborate is added to the solution (Saferstein 2001).

41. DETECTION OF SEMEN STAINS
 Semen stains can be characterized with visualization of sperm cells, acid
phosphatase (AP) or prostate specific antigen (PSA or p30) tests.
 A microscopic examination to look for the presence of spermatozoa is
performed in some laboratories on sexual assault evidence. But two issues
exist…
 Aspermic or oligospermic males have either no sperm or a low sperm count in
their seminal fluid ejaculate.
 Vasectomized males will not release sperm.

42. Acid phosphatase Detection
 Acid phosphatase is an enzyme secreted by the prostate gland into seminal
fluid and found in concentrations up to 400 times greater in semen than in
other body fluids (Sensabaugh 1979, Saferstein 2001). A purple color with the
addition of a few drops of sodium alpha naphthylphosphate and Fast Blue B
solution or the fluorescence of 4-methyl umbelliferyl phosphate under a UV
light indicates the presence of AP.
 Prostate specific antigen was discovered in the 1970s and shown to have
forensic value with the identity of a protein named p30 due to its apparent
30000 molecular weight (Sensabaugh 1978).

43. DIRECT OBSERVATION OF SPERM
 A common method of doing this is to recover dried semen evidence from
fabric or on human skin with a deionized water-moistened swab.
 A portion of the recovered cells are then placed onto a microscope slide and
fixed to the slide with heat. The immobilized cells are stained with a
„Christmas Tree‟ stain.
 „Christmas Tree‟ stain consists of aluminum sulfate, nuclear fast red, picric
acid, and indigo carmine (Shaler 2002).
 The stained slide is then examined under a light microscope for sperm cells
with their characteristic head and long tail. The Christmas Tree stain marks
the anterior sperm heads light red or pink, the posterior heads dark red, the
spermatozoa‟s mid-piece blue, and the tails stain yellowish green (Shaler
2002).

44. DIRECT CAPTURE OF SPERM CELLS
 Laser-capture microdissection, which is commonly used to select tumor cells
from surrounding tissue on microscope slides. Sperm cells from sexual assault
evidence spread on microscope slides can be collected with laser-capture
microdissection to perform reliable STR testing (Elliot et al. 2003).
 When sperm cells are observed in the field of view of the microscope, a tiny
laser is activated and a thin plastic film placed over the slide melts at the
specific point of laser light contact to capture the cell of interest. By moving
the microscope slide around, dozens of sperm cells are collected onto this
thin film that sits directly above the sample. The collection film is then
transferred to a tube where DNA from the isolated sperm can be extracted
and amplified using the polymerase chain reaction

45. DIRECT CAPTURE OF SPERM CELLS
 Sperm cells can be collected on magnetic particles or beads that can be
coated with antibodies specific for sperm proteins (Marshall 2002). The beads
are then washed to remove the female epithelial cells. Finally, the purified
sperm are placed into a PCR reaction to produce a DNA profile of the
perpetrator.
 This approach depends on sperm being intact, which is not always the case
with old sexual assault evidence.

46. DETECTION OF SALIVA STAINS
 A presumptive test for amylase is used for indicating the presence of saliva,
which is especially difficult to see since saliva stains are nearly invisible to
the naked eye
 Saliva stains may be found on bite-marks, cigarette butts, and drinking
vessels (Abaz et al. 2002, Shaler 2002).
 Two common methods for estimating amylase levels in forensic samples
include
 Phadebas test
 Starch iodine radial diffusion test

47. DNA EXTRACTION
 A biological sample obtained from a crime scene a paternity case contains a
number of substances besides DNA.
 Cellular proteins that package and protect DNA in the environment of the cell
can inhibit the ability to analyze the DNA. So, DNA molecules must be
separated from other cellular material before they can be examined.
 There are three DNA extraction methods
 Organic extraction
 Chelex extraction
 FTA paper

48. Organic extraction or phenol chloroform
extraction
 Organic extraction used for situations where either RFLP or PCR typing is
performed.
 High molecular weight DNA, which is essential for RFLP methods, may be
obtained most effectively with organic extraction.

49. Organic Extraction Procedure
Blood
stain
SDS, DTT, EDTA
and
proteinase K
Phenol,
chloroform,
isoamyl
alcohol
QUANTITATE
DNA
PERFORM
PCR
INCUBATE (56 oC)
Centrifuge
VORTEX
Centrifuge
CONCENTRATE sample
(Centricon/Microcon-100 or ethanol precipitation)
Centrifuge
TRANSFER aqueous
(upper) phase to new tube
TE
buffer

50. Chelex method
 The Chelex method of DNA extraction is more rapid than the organic
extraction method.
 Chelex extraction involves fewer steps and thus fewer opportunities for
sample-to-sample contamination.
 However, it produces single stranded DNA as a result of the extraction process
and therefore is only useful for PCR-based testing procedures.

51. Chelex Extraction
Blood
stain Water INCUBATE
(ambient)
5%
Chelex
INCUBATE (100 oC)
REMOVE
supernatant
INCUBATE (56 oC)
QUANTITATE
DNA
PERFORM
PCR
Centrifuge
Centrifuge

52. FTA Paper Extraction
 FTA paper was developed by Lee Burgoyne at Flinders University in Australia
as a method for storage of DNA (Burgoyne et al. 1994).
 FTA paper is an absorbent cellulose-based paper that contains four chemical
sub- stances to protect DNA molecules from nuclease degradation and
preserve the paper from bacterial growth (Burgoyne 1996).
 DNA on FTA paper is stable at room temperature over a period of several
years.

53. Procedure
 Add a spot of blood to the paper and allowing the stain to dry. The cells are
lysed upon contact with the paper and DNA from the white blood cells is
immobilized within the matrix of the paper.
 A small punch of the paper is removed from the FTA card bloodstain and
placed into a tube for washing. The bound DNA can then be purified by
washing it with a solvent to remove heme and other inhibitors of the PCR
reaction.
 This purification of the paper punch can be seen visually because as the
paper is washed, the red color is removed with the supernatant.
 The clean punch is then added directly to the PCR reaction. Alternatively,
some groups have performed a Chelex extraction on the FTA paper punch and
used the supernatant in the PCR reaction (Lorente et al. 1998, Kline et al.
2002).

54. FTA Paper
WASH Multiple Times with
extraction buffer
PCR
Reagents
Apply blood to
paper and allow
stain to dry
PUNCH
REMOVE
supernatant
PERFORM
PCR
(NO DNA QUANTITATION
REQUIRED)

55. Storing DNA
 Extracted DNA is typically stored at −20°C, or even −80°C for long-term stor-
age, to prevent nuclease activity.
 Nucleases are enzymes (proteins) found in cells that degrade DNA to allow
for recycling of the nucleotide components.
 Nucleases need magnesium to work properly so one of the measures to
prevent them from digesting DNA in blood is the use of purple-topped tubes
containing a blood preservative known as EDTA.
 The EDTA chelates, or binds up, all of the free magnesium and thus prevents
the nucleases from destroying the DNA in the collected blood sample.

56. DIFFERENTIAL EXTRACTION
 Differential extraction is a modified version of the organic extraction method
that separates epithelial and sperm cells.
 Differential extraction was first described in 1985 (Gill et al. 1985).
 Now commonly used by the FBI Laboratory and other forensic crime
laboratories to isolate the female and male fractions in sexual assault cases
that contain a mixture of male and female DNA.

57. Perpetrator‟s sperm
mixed with victim‟s
epithelial cells
Centrifug
e
REMOVE
supernatant
SDS, EDTA and
proteinase K
(cell lysis buffer)
Remove a
portion of
the mixed
stain
SDS, EDTA and
proteinase K + DTT
Incubate
at 37 oC
sperm
pellet
DTT
lyses
sperm
heads
“Male Fraction” “Female Fraction”sperm
pellet

58. SOLID-PHASE EXTRACTION
 Two approaches
 QIAamp spin columns
 DNA IQ™ system

59. QIAamp spin columns
 In this approach nucleic acids selectively absorb to a silica support, such as
small glass beads, in the presence of high concentrations of chaotropic salts
such as guanidine hydrochloride, guanidine isothiocyanate, sodium iodide,
and sodium perchlorate (Vogelstein and Gillespie 1979, Boom et al. 1990,
Duncan et al. 2003).
 These chaotropic salts disrupt hydrogen bonding networks in liquid water and
thereby make denatured proteins and nucleic acids more thermodynamically
stable than their correctly folded or structured counter- parts (Tereba et al.
2004).
 If the solution pH is less than 7.5, DNA adsorption to the silica is typically
around 95% and unwanted impurities can be washed away.
 Under alkaline conditions and low salt concentrations, the DNA will efficiently
elute from the silica material.

60. DNA IQ™ system
 The DNA IQ™ system utilizes the same silica-based DNA binding and elution
chemistries as QIAGEN kits but with silica-coated paramagnetic resin (Tereba
et al. 2004).
 With this approach, DNA isolation can be performed in a single tube by simply
adding and removing solutions.
 First, the DNA molecules are reversibly bound to the magnetic beads in
solution with a solution pH of less than 7.5. A magnet is used to draw the
silica- coated magnetic beads to the bottom or side of the tube leaving any
impurities in solution. These solution impurities (proteins, cell debris, etc.,)
can easily be removed by drawing the liquid off of the beads. The magnetic
particles with DNA attached can be washed multiple times to more thoroughly
clean the DNA. Finally, a defined amount of DNA can be released into solution
via heating for a few minutes.

61. OTHER METHODS FOR DNA EXTRACTION
 A simple closed tube DNA extraction method has been demonstrated with a
thermal stable protease that looks very promising (Moss et al. 2003).
 Microwave extraction has been used to shorten the conventional organic
extraction method by several hours and to yield genomic DNA that could be
PCR-amplified (Lee et al. 1994).
 The addition of 6M NaCl to a proteinase K-digested cell extract followed by
vigorous shaking and centrifugation results in a simple precipitation of the
proteins (Miller et al. 1988). The supernatant containing the DNA portion of
cell extract can then be added to a PCR reaction.
 A simple alkaline lysis with 0.2M NaOH for five minutes at room temperature
has been shown to work as well (Rudbeck and Dissing 1998, Klintschar and
Neuhuber 2000).

62. PCR INHIBITORS AND DNA DEGRADATION
 The presence of inhibitors or degraded DNA can manifest themselves by
complete PCR amplification failure or a reduced sensitivity of detection
usually for the larger PCR products.
 Two PCR inhibitors commonly found in forensic cases are hemoglobin and
indigo dyes from denim.
 Melanin found in hair samples can be a source of PCR inhibition when trying
to amplify mitochondrial DNA.
 These inhibitors likely bind in the active site of the TaqDNA polymerase and
prevent its proper functioning during PCR amplification.
 Heat and humidity, which speed up hydrolytic cleavage, are enemies of intact
DNA molecules. UV irradiation (e.g., direct sunlight) can lead to cross-linking
of adjacent thymine nucleotides on the DNA molecule, which will prevent
passage of the polymerase during PCR.

63. DNA QUANTITATION
 Early methods for DNA quantitation typically involved either absorbance at a
wavelength of 260nm or fluorescence after staining a yield gel with ethidium
bromide.
 Unfortunately, because these approaches are not very sensitive, they
consume valuable forensic specimens that are irreplaceable. In addition,
absorbance measurements are not specific for DNA and contaminating
proteins or phenol left over from the extraction procedure can give falsely
high signals.
 Modern techniques are
 Slot blot procedure
 Fluorescence- based microtiter plate assays („real-time or quantitative PCR‟)

64. Slot Blot
 Slot blot procedure test is specific for human and other primate DNA due to a
40 base pair (bp) probe that is complementary to a primate-specific alpha
satellite DNA sequence D17Z1 located on chromosome 17 (Waye et al. 1989,
Walsh et al. 1992).
 The slot blot assay was first described with radioactive probes (Waye et al.
1989) but has since been modified and commercialized with
chemiluminescent or colorimetric detection formats (Walsh et al. 1992).
 Slot blots is fairly sensitive as it can detect both single-stranded and double-
stranded DNA down to levels of approximately 150pg or about 50 copies of
human genomic DNA. A 150pg DNA standard can be detected after only a 15
minutes exposure to X-ray film (Walsh et al. 1992).

65. Cont…
 Slot blots involve the capture of genomic DNA
on a nylon membrane followed by addition of a
human-specific probe. Chemiluminescent or
colorimetric signal intensities are compared
between a set of standards and the samples.
 Slot blot quantitation is a relative
measurement involving the comparison of
unknown samples to a set of standards that are
prepared usually via a serial dilution from a DNA
sample of known concentration.
 While comparison of the digital capture and
quantification of slot blot images has been
demonstrated with a charged-coupled device
(CCD) camera imaging system (Budowle et al.
2001).
20 ng
10 ng
5 ng
2.5 ng
1.25 ng
0.63 ng20 ng
10 ng
5 ng
2.5 ng
1.25 ng
0.63 ng
Calibration
standards
Calibration
standards
Unknown Samples
~2.5 ng

66. PICOGREEN MICROTITER PLATE ASSAY
 The Forensic Science Service has developed a PicoGreen assay that is capable
of detecting as little as 0.25ng/mL of double-stranded DNA in a 96-well
microtiter plate format (Hopwood et al. 1997).
 PicoGreen is a fluorescent interchelating dye whose fluorescence is greatly
enhanced when bound to double-stranded DNA.
 To perform this microtiter plate assay, 5µL of sample are added to 195µL of a
solution containing the PicoGreen dye. Each sample is placed into an
individual well on a 96-well plate and then examined with a fluorometer. A
96-well plate containing 80 individual samples and 16 calibration samples can
be analyzed in under 30 minutes (Hopwood et al. 1997).
 Unfortunately, this assay quantifies total DNA in a sample and is not specific
for human DNA.

67. ALUQUANT™ HUMAN DNA QUANTITATION
SYSTEM
 The Promega Corporation has developed a human DNA quantitation system
that enables sensitive detection of DNA (Mandrekar et al. 2001). The
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
oxidation of luciferin with production of light. The light intensity is read by a
luminometer and is proportional to the amount of DNA present in the sample.
 The AluQuant™ assay possesses a range of 0.1–50ng for human DNA and can be
automated on a robotic liquid handling workstation (Hayn et al. 2004)

68. END-POINT PCR FOR DNA QUANTIFICATION
 A approach for testing the „amplifiability‟ of a DNA sample is to perform an
end-point PCR test. In this approach a single STR locus (Kihlgren et al. 1998,
Fox et al. 2003) or other region of the human genome, such as an Alu repeat
(Sifis et al. 2002, Nicklas and Buel 2003a), is amplified along with DNA
samples of known concentrations.
 A standard curve can be generated from the samples with known amounts to
which samples of unknown concentration are compared. A fluorescent
intercalating dye such as SYBR® Green can be used to detect the generated
PCR products.
 Based on the signal intensities resulting from amplification of the single STR
marker or Alu repeat region, the level of DNA can be adjusted prior to
amplifying the multiplex set of DNA markers in order to obtain the optimal
results.

69. Calculation of the quantity of DNA in a cell
1. Molecular Weight of a DNA Basepair = 618g/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
2. Molecular weight of DNA = 1.85 x1012 g/mol
There are 3 billion base pairs in a haploid cell ~3 x 109 bp
(~3 x 109 bp) x (618 g/mol/bp) = 1.85 x 1012 g/mol
3. Quantity of DNA in a haploid cell = 3 picograms
1 mole = 6.02 x 1023 molecules
(1.85 x 1012 g/mol) x (1 mole/6.02 x 1023 molecules)
= 3.08 x 10-12 g = 3.08 picograms (pg)
A diploid human cell contains ~6 pg genomic DNA
4. One ng of DNA contains the DNA from 167 diploid cells
1 ng genomic DNA (1000 pg)/6pg/cell = ~333 copies of each locus (2 per 167 diploid genomes)

70. Chapter 4
The Polymerase Chain Reaction
(DNA Amplification)

71. PCR Process
 First described in 1985 by Kary
Mullis and members of the Human
Genetics group at the Cetus
Corporation (now Roche Molecular
Systems), PCR has revolutionized
molecular biology with the ability
to make millions of copies of a
specific sequence of DNA in a
matter of only a few hours.
 This molecular process involves
heating and cooling samples in a
precise thermal cycling pattern
over ~30 cycles.
94 oC
60 oC
72 oC
Time
Temperature
Single Cycle
Typically 25-35 cycles
performed during PCR
94 oC 94 oC 94 oC
60 oC60 oC
72 oC72 oC
The denaturation time in the first
cycle is lengthened to ~10 minutes
when using AmpliTaq Gold to
perform a “hot-start” PCR

72. Separate
strands
(denature)
Add primers
(anneal)
Make copies
(extend primers)
Repeat Cycle,
Copying DNA
Exponentially
Starting DNA
Template
5’
5’
3’
3’
5’
5’
5’
3’ 3’
3’3’5’
Forward primer
Reverse primer

73. PCR
 PCR is commonly performed with a sample volume in the range of 5–100µL.
At such low volumes, evaporation can be a problem and accurate pipetting of
the reaction components can become a challenge.
 On the other hand, larger solution volumes lead to thermal equilibrium issues
for the reaction mixture because it takes longer for an external temperature
change to be transmitted to the center of a larger solution than a smaller
one.
 Therefore, longer hold times are needed at each temperature, which leads to
longer overall thermal cycling times. Most molecular biology protocols for PCR
are thus in the 20–50µL range.

74. PCR COMPONENTS
 The most important components of a PCR reaction are the two primers, which
are short DNA sequences that precede or „flank‟ the region to be copied.
 The other components of a PCR reaction consist of
 Template DNA that will be copied.
 Building blocks made up of each of the four nucleotides
 DNA polymerase that adds the building blocks in the proper order based on
the template DNA sequence.

75. CONTROLS USED TO MONITOR PCR
 A „negative control‟, which is the entire PCR reaction mixture without any
DNA template. The negative control usually contains water or buffer of the
same volume as the DNA template, and is useful to assess whether or not any
of the PCR components have been contaminated by DNA (e.g., someone else
in your lab).
 A „positive control‟ is a valuable indicator of whether or not any of the PCR
components have failed or were not added during the reaction setup phase of
experiments conducted. The DNA template should be amplified with the same
PCR primers as used on the rest of the samples in the batch that is being
amplified. The purpose of a positive control is to ensure confidence that the
reaction components and thermal cycling parameters are working for
amplifying a specific region of DNA.

76. Whole Genome Amplification (WGA)
 WGA involves a different DNA polymerase than the TaqGold enzyme commonly
used in forensic DNA analysis. WGA amplifies the entire genome using random
hexamers as priming points.
 The WGA enzymes work by multiple displacement amplification (MDA), which
is sometimes referred to as rolling circle amplification. MDA is isothermal with
an incubation temperature of 30°C and requires no heating and cooling like
PCR.

77. THERMAL CYCLERS
 The instrument that heats and cools a DNA sample in order to perform the
PCR reaction is known as a thermal cycler. Precise and accurate sample
heating and cooling is crucial to PCR in order to guarantee consistent results.
 The most prevalent thermal cycler in forensic DNA laboratories is the
GeneAmp® PCR System 9600 from Applied Biosystems (Foster City, CA). The
„9600‟ can heat and cool 96 samples in an 8 ×12-well microplate format at a
rate of approximately 1°C per second.

78. Hot Start PCR
 At a low temperature for the primers to bind to each other creating products
called „primer dimers.‟ These are a particular problem because their small
size relative to the PCR products means that they will be preferentially
amplified.
 Low-temperature mispriming can be avoided by initiating PCR at an elevated
temperature, a process usually referred to as „hot start‟ PCR. Hot start PCR
may be performed by introducing a critical reaction component, such as the
polymerase, after the temperature of the sample has been raised above the
desired annealing temperature (e.g., 60°C).
 A more important disadvantage is the fact that the sample tubes must be
opened at the thermal cycler to introduce the essential component, which
gives rise to a greater opportunity for cross-contamination between samples.

79. AMPLITAQ GOLD DNA POLYMERASE
 AmpliTaq Gold™ DNA polymerase is a chemically modified enzyme that is
rendered inactive until heated (Birch et al. 1996). An extended pre-
incubation of 95°C, usually for 10 or 11 minutes, is used to activate the
AmpliTaq Gold.
 AmpliTaq Gold is not compatible with the pH 9.0 buffers used for regular
AmpliTaq DNA polymerases (Moretti et al. 1998). This fact is because the pH
does not get low enough to remove the chemical modifications on TaqGold
and thus the enzyme remains inactive. Tris buffers with a pH 8.0 or 8.3 at
25°C work the best with TaqGold.

80. MULTIPLEX PCR
 The PCR permits more than one region to be copied simultaneously by simply
adding more than one primer set to the reaction mixture (Edwards and Gibbs
1994). The simultaneous amplification of two or more regions of DNA is
commonly known as multiplexing or multiplex PCR.
 For a multiplex reaction to work properly the primer pairs need to be
compatible. The primer annealing temperatures should be similar and
excessive regions of complementarity should be avoided to prevent the
formation of primer-dimers that will cause the primers to bind to one another
instead of the template DNA.

81. REAL-TIME (QUANTITATIVE) PCR
 Real-time PCR, which was first described by Higuchi and co-workers at the
Cetus Corporation in the early 1990s (Higuchi et al. 1992, Higuchi et al. 1993), is
sometimes referred to as quantitative PCR or „kinetic analysis‟ because it
analyzes the cycle-to-cycle change in fluorescence signal resulting from
amplification of a target sequence during PCR.
 This analysis is performed without opening the PCR tube and therefore can
be referred as a closed-tube or homogeneous detection assay.
 The most common approaches utilize either the fluorogenic 5′ nuclease assay
better known as TaqMan®.

82. THE 5′ NUCLEASE ASSAY (TAQMAN)
 TaqMan probes are labeled with two fluorescent dyes that emit at different
wavelengths. The probe sequence is intended to hybridize specifically in the
DNA target region of interest between the two PCR primers (Ong and Irvine
2002). Typically the probe is designed to have a slightly higher annealing
temperature compared to the PCR primers so that the probe will be
hybridized when extension (polymerization) of the primers begins
 The „reporter‟ (R) dye is attached at the 5′-end of the probe sequence while
the „quencher‟ (Q) dye is synthesized on the 3′-end. A popular combination of
dyes is FAM or VIC for the reporter dye and TAMRA for the quencher dye.
 When the probe is intact and the reporter dye is in close proximity to the
quencher dye, little-to-no fluorescence will result because of suppression of
the reporter fluorescence due to an energy transfer between the two dyes.

83. Cont…
 The Taq DNA polymerase used has a 5′-exonuclease activity and therefore will
begin to chew away at any sequences in its path (i.e., those probes that have
annealed to the target sequence). When the reporter dye molecule is
released from the probe and it is no longer in close proximity to the quencher
dye, it can begin to fluoresce.

84. Polymerization and
Strand Displacement
R
Q
Forward
primer
Reverse
primer
3‟
5‟ 3‟
5‟
3‟5‟
5‟
5‟
Forward
primer
Reverse
primer
3‟
5‟ 3‟
5‟
5‟
5‟
Q
R
3‟ Probe Cleavage
(release of reporter
dye)
Forward
primer
Reverse
primer
3‟
5‟ 3‟
5‟
5‟
5‟
Q
Completion of
Polymerization
TaqMan
probe
Fluorescence occurs when
reporter dye and quencher dye
are no longer in close proximity

85. ADVANTAGES OF PCR WITH FORENSIC
SPECIMENS
 Very small 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.

86. DISADVANTAGES OF PCR WITH FORENSIC
SPECIMENS
 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.