JS 111: Introduction to Genetics and DNA
The Scientific Basis of DNA Typing
I.
Pre class activities
a.
b.
II.
Quiz
Assignment and announcements
Learning Objectives
a.
b.
Understand why scientists study DNA.
Introduction to Basic Genetics- Heredity
Be able to define: cell, nucleus, chromosome,
genetic linkage, alleles, homozygous vs
heterozygous, independent segregation and random
assortment
Be able to draw a Punnett Square to illustrate allele
inheritance
c.
Introduction to DNA
a.
Understand the Basic Facts and Function of DNA
b.
Be able to draw DNA structure – Base Pairing AT, GC
c.
Understand DNA replication
d.
Overview of Methods Used to study DNA
a.
Screening, Extraction, Quantification, Typing RFLP v
PCR, Interpretation
Assignments and Announcements
• See handout
• Study for exam 1
Who Cares?
•
Law Enforcement
– Criminal Investigation- Casework, Databanks
– Reuniting immigrant families- Paternity
– Missing persons
•
Evolutionary, Agricultural and Zoological applications
– Assessing genetic diversity
– Fingerprinting endangered species and pathogens
– Assessing unrelatedness to breed for increasing genetic diversity
– Assessing relationships for all biological predictions
– Ancient DNA analyses for reconstructing history (how we populated the globe)
•
Other Human Applications
– Making sense of the Human Genome project results- Bioinformatics
– Developing rapid medical diagnostics such as those associated with triplet
repeat diseases (STRs)- (Moxon et al. 1999 Sci Amer. 280:94)
– Understanding the molecular basis of development, disease and aging
– Screening candidates for bone marrow/organ transplants and grafts
WE ALL DO!
Steps in Forensic DNA typing
Evaluation- Is it there?
(Figure 6.1 Rudin and Inman 2001)
1.
Start with biological sample
2.
Screen- blood? Semen? Saliva, human?
Extraction- Get and clean DNA
3.
Open cells  Get DNA
4.
Methods to get DNA and purify DNA
Quantify- Determine quality and quantity?
5.
QuantifyHow good and how much did you get?
Type to determine and compare alleles
6. RFLP vs PCR
7. Determine alleles and compare DNA types
Or alleles present in samples and references
Interpretation of Results
DNA Facts and Jargon
Where is it? How is it stored?
DNA is found in every
*cell= basic unit of life
Inside nuclei (organization center for the cell) and mitochondria (ATP powerhouse of the
cell) & chloroplasts for plants- (making our food via photosynthesis)
Nuclei are not found in red blood cells
In white blood cells, saliva, skin, hair fingernails, urine, feces, vomitus, earwax etc.
DNA in the Cell
In nuclei, mitochondria and chloroplasts (plants) organized in
chromosomes (wound around histones)
“DNA double bagging”
chromosome
cell nucleus
Double stranded
DNA molecule
Target Region for PCR
Individual
nucleotides
I. Intro to DNA : Facts and Jargon
DNA: Deoxyribonucleic acid
Different in every *individual
The same in every **cell of an
individual's body
*except for identical twins that have the same DNA "The time honored
method of cloning humans"
** diseased individuals may be mosaics
DNA function
What’s it do?
DeoxyriboNucleic Acid : blueprints of life
Replication, Information Storage and Mutation
Central Dogma
information flow--------------->
DNA------->RNA------>protein
transcription
translation
DNA Organization and Inheritance
Human Genome Contains
23 Pairs of Chromosomes
http://www.ncbi.nlm.nih.gov/genome/guide/
It is inherited from your mom and dad
1
2
3
4
5
6
7
8
9
10 11 12
13 14 15 16 17 18 19 20 21 22 X
Y
Sex-chromosomes
Basic Chromosome Structure
and Nomenclature
Chromosome 12
Chromosome 12
telomere
p
P= short arm
Band 3
12p3
(short arm)
Q= long arm
Center= Centromere (involved in
mitosis and meiosis attachment to
spindle fibers)
centromere
q
End= Telomere (involved in aging)
(long arm)
Band 5
12q5
D12S459
telomere
D= DNA
12= Chromosome 12
S=Single copy sequence
459= 459th locus described on C12
Definitions of Locus and Allele
• 2 pairs of Homologous
chromosomes (white from dad,
dark from mom)
• Locus (singular) or Loci
(plural) are defined locations
where specific genes or markers
are found
• Alleles are different forms of
the same gene or marker
• When alleles have the same
form on a locus they are said to
be homozygous. When
different they are heterozygous
Where’s Daddy?
PCR product size (bp)
11
12
8
14
14
11 12
8
14
12
Review- Mendelian Genetics
Law of Independent SegregationBig D and little d will evenly segregate into the next generation
And results in equal inheritance from mom and dad
Lee family pedigree
Hair color
D = Dark
d = blonde
Name
Steve
Julie
Genotype
D,D
d,d
•d
Gametes possible
D
d
d
•D •Dd Dd
•D •Dd Dd
Julie
Steve
Punnett Square
Gabriel
Sierra
Sierra
grandkid 1
Future mate
grandkid 2
Inheritance Review
Dad is homozygous (A,
A) and mom is
heterozygous (A, a).
In your teams, draw the
Punnett Sqaure that
demonstrates the
inheritance of these
alleles.
What percentage of their
children will be
heterozygous?
Review- Mendelian Genetics
Law of Random Assortment
• This law states that markers on different chromosomes are
generally inherited independently of one another and are not
inherited together more often than might be expected by chance.
• Those displaying random assortment are said to be in linkage
equilibrium.
• Those that show genetic linkage such as those located close
together on the same chromosome are said to be in linkage
disequilibrium and are found more often together than expected
by chance. Example is blonde hair and blue eyes.
• Markers that are in linkage equilibrium are desirable for forensic
DNA. The product rule can be applied to those displaying
random assortment thus resulting in a higher power of
discrimination than those that are linked.
DNA Structure
What is it?
Bases (AGCT) form the stairs of the ladder, are
faithfully paired and exhibit
differences.
P
S-A : T-S
P
P
S-G : C-S
P
P
S-A : T-S
P
P
S-G : C-S
P
Hydrogen bonds
3’
5’
5’
A=T
GC
A=T
GC
hybridized
strands
3’
T=A
Phosphate-sugar backbone
T=A
CG
CG
A=T
A
T
G
C
G
CG
CG
G
C
3’
A=T
T=A
A
T
denatured
strands
C
5’
3’
GC
Sugars (S) and phosphates (P) form the sides of the ladder (identical for all
DNA).
DNA Structure
• Primary genetic material
is composed of two
complementary strands
• Form a double helix or
twisted ladder
• Sides are sugar phosphate
and the steps are base
pairs
• Four Bases- 2 Purines –
Adenine and Guanine and
2 Pyrimidines- Cytosine
and Thymine
• Asian Guys are Pure!
DNA Structure
Nucleotides are the building blocks themselves
composed of PBS
Nucleotides-PBS
Phosphate (negative charge)
Base (AGCT-Asian Guys Can Teach)
Sugar (deoxyribose-5C)
Phosphate-Sugars
Connected by phosphodiester linkages
Basic Components of Nucleic
Acids
Phosphate
5’end
|
Phosphate
|
Sugar—Base…
|
Phosphate
|
Sugar—Base…
|
3’end
Base
Base
(A, T, C, or G)
O
5’
HO
P
O
O-
Sugar
CH 2
O
H
H
H 3’
O
2’
4’
1’
H
Base
(A, T, C, or G)
H
5’
HO
P
O-
O
CH 2
O
4’
H
1’
H
H 3’
2’
OH H
H
DNA Structure
2 Complimentary,
Antiparallel Strands
held together by Base Pairs- H Bonds
A:T held with 2 H Bonds
.
G:C held with 3 H Bonds
Polymerase Chain Reaction:
PCR is simply repeated rounds of DNA replication
PCR based systems are rapid,
require less material than RFLP
and less time for typing
• Molecular xeroxing
• Calvin and Hobbes example
Replication of DNA is Semi Conservative
One old and one new
http://dir.niehs.nih.gov/dirlmg/repl.html
Enzymes of Replication
DNA is replicated or copied in our cells.
When completed, the new double strands
consist of one old template and one newly
made strand- This is called semi
conservative replication.
There are many enzymes that are required.
They include unwinding (helicases,
gyrases), priming (primases), copying (DNA
polymerases) and touch up enzymes (DNA
ligases).
DNA Replication
5-P’
•5 required ingredients (components)- primer,
template, Mg, dntps, DNA polymerasePTMDD-(please to make DNA doubled)
Primer
5’
HO
P
O-
3-OH’
O
•DNA Polymerase catalyzes the template
directed (A-T, G-C), incorporation of dNTPs
(PP is released) forming a 3’-5’ phosphodiester
linkage
Base
(A, T, C, or G)
O
CH 2
O
H
H
H 3’
O
2’
4’
1’
• Direction of synthesis 5’3’ using primer
3’OH to attach incoming nucleotide
H
Base
(A, T, C, or G)
H
5’
HO
P
O-
O
CH 2
O
4’
H
1’
H
H 3’
2’
OH H
3-OH’
P-P-HO
Mg++
Template = Old
dNTP Base
O
H
DNA polymerase
P
O-
O
CH 2
O
H
4’
1’
H
H 3’
2’
OH H
O
H
5’
HO
5-P’
(A, T, C, or G)
5’
P
O-
O
CH 2
O
DNA St. Patricks Day Salute to the Molecule of Heredity
From Biology 110- UNC 1993 Steve Lee
The molecular structure today
Is heredity’s DNA
With nucleotides
completely comprised
of a sugar and phosphate and base
Adenine and thymine can base pair
Forming two hydrogen bonds for one stair
Cytosine and guanine
pair with three in between
and are equal in size when compared
The bases you see are so keen
They include thymine and adenine
Cytosine and one more
with guanine can store
all the info with rungs in between
DNA strands are just not the same
One is coding and one is called lame (anticoding)
They are opposite
in direction and this
is called antiparallel in name
The sides of the ladder you know,
are sugar and phosphate which show
that Franklin was right
double helix is tight
ten base pairs per turn in a row
Complimentary nature of strands
lets replication proceed just as planned
with A paring to T
and G pairing to C
the fidelity is precise and quite grand
Steps in Forensic DNA typing
Evaluation- Is it there?
(Figure 6.1 Rudin and Inman 2001)
1.
Start with biological sample
2.
Screen- blood? Semen? Saliva, human?
Extraction- Get and clean DNA
3.
Open cells  Get DNA
4.
Methods to get DNA and purify DNA
Quantify- Determine quality and quantity?
5.
QuantifyHow good and how much did you get?
Type to determine and compare alleles
6. RFLP vs PCR
7. Determine alleles and compare DNA types
Or alleles present in samples and references
Interpretation of Results
Review: DNA is organized
inside the cell nucleus and mitochondria
DNA Extraction
After screening tests are
performed, a spot of the
material containing the
biological sample is cut
and placed into a tube.
In one type of extraction
method (organic), heat and
chemicals are added, and
protein is removed.
Then the pure DNA is
recovered by filtration in
which the non-DNA
material goes through a
sieve.
(analogous to a collection
of your pasta in a colander)
Different DNA Extraction Methods
The organic method
generally yields the highest
quantity and quality DNA. The
main disadvantages are that it is
tedious with lots of steps and
utilizes corrosive chemicals.
The Chelex method is used
when the sample contains very
few cells and the reduced
number of handling steps is the
primary advantage. The main
disadvantage is that it is yields
crude DNA that is not as pure
FTA paper is used to collect
reference samples. It can be
stored at room temperature,
requires minimal handling and
no quantification is required.
Differential Extraction Method
For Sexual Assault Evidence
Female
cell
Spermatozoa
• Isolation of DNA from mixtures of
cells in sexual assault evidence
• Based on differences in cell
membranes
– Spermatozoa membranes have
special cross links (sulphursulphur bonds)
– These membranes are quite
resistant to opening.
– Vaginal epithelial cells do not
contain these membranes and
are more easily broken open
Sperm and v cell
mixture
Lysis- open
v cell extract
Female DNA
Female DNA
Lysis- open
sperm extract
Male DNA
Male DNA
Quantification of DNA
• Following extraction, the next step is to
determine the quantity of the DNA
• DNA typing methods RFLP and PCR
require different amounts and different
quality of DNA.
• RFLP typically required 50ng. PCR
typically requires less than 0.5ng to 1 ng:
100 times less!
Quantification of DNA using
Gel Electrophoresis
• Total DNA can be quantified by
running the samples in a gel.
• Typically, gels are made up of agarose
(a carbohydrate from seaweed).
• Known DNA quantities are included
• Samples are then subject to an electric
current and is called electrophoresis.
• DNA is negatively charged and will
migrate toward the positive electrode…
• Comparisons of the results are done
visually or with computer software to
determine the amount of DNA in the
unknown sample.
(-)
L K K – u u u
wells
Intact
DNA
Degraded
DNA
(+)
Direction of DNA fragment movement
Smaller fragments move faster and are found
Near the bottom of the gel
Slot Blot quantification :
DNA-DNA Hybridization
DNA is where it’s AT
• DNA samples may contain non human DNA
• In order to quantify the amount of human
DNA in a sample, a human specific test is
required
• One such test is DNA-DNA hybridization
using a human specific probe: D17Z1
Slot blot hybridization
• Like in yield gels, known amounts of DNA (human) are
included
• DNA hybridization of D17Z1 will occur only if the sample
contains human DNA
• Detection of the hybridized fragments is done using an
enzyme linked assay- yielding light or color
Real Time PCR
Quantitative PCR- QPCR
http://pathmicro.med.sc.edu/RTPCR/rt-pcr.ppt
• Real-time QPCR has several advantages over the other methods in
that it is extremely accurate and sensitive over a broad dynamic
range, and it occurs in a closed-tube system, reducing the potential
for carryover contamination.
• Using this technique, a forensic biologist can monitor and quantify
the accumulation of PCR products during log phase amplification.
(Heid et al., 1996).
• Several RT PCR human specific assays are now available that target
autosomal, Alu repeats, Y chromosome and mtDNA (Andréasson et
al. 2002, Nicklas et al. 2003, Green et al. 2005, Andréasson et al.
2006, Horsman et al. 2006).
• The assays may be performed on single targets or in multiplexes
(Timken et al. 2005, Walker et al. 2005, Nicklas et al. 2006).
• Recently, the detection of degraded vs intact human DNA and PCR
inhibitors has been reported (Swango et al. 2006).
RNA based quantification methods
• Different genetic expression patterns (mRNAs) exist in different tissue
types.
• Body fluid identification has been reported based on their mRNA
profiles (Juusola and Ballantyne 2003 and 2005, Nussbaumer et al.
2006)
• In addition, the age of a bloodstain was reported using analysis of
mRNA: rRNA ratios (Anderson et al. 2005). This information may be
useful in establishing the time of the crime.
• Advantages of the mRNA-based approach, versus the conventional
biochemical tests, include greater specificity, simultaneous and semiautomatic analysis, rapid detection, decreased sample consumption
and compatibility with DNA extraction methodologies.
• The quantification of the amounts of the mRNA species relative to
housekeeping genes is a critical aspect of the assays (Juusola and
Ballantyne 2003).
Comparison of Methods used for
DNA Quantification
Method
UV Spectrophotometry
Yield Gel electrophoresis
Slot Blot
Yield Gel blot
Pico-green microtitre plate
Ease
+++++
+++
++
+
++++
Cost
+
+
++
++
++
Sensitivity
++
+
+++
+++
++++
Result
Total DNA
Int vs deg DNA
Human DNA
Int. vs. deg human DNA
Total DNA
Alu Quant
Real time PCR assays
Real time PCR assays
+++
+++
+++
+++
+++
+++
++++
+++++
+++++
Human DNA
Human DNA
Int.vs. deg.Human DNA
Summary 1 (from last week)
• Why study DNA
– Law enforcement, evolution, agricultural, and human
applications-medical diagnostics
• DNA Biology and Genetics
– DNA is contained in cells –the basic unit of life
– Found in nuclei, mitochondria and chloroplasts
– Organized in chromosomes. Located at positions called loci and
come in different forms or alleles.
– Homozygous if the same, heterozygous if different
– Alleles segregate independently and assort randomly when on
different chromosomes. Random assortment is desired for forensic
DNA loci.
• DNA Function and Structure
– DeoxyriboNucleic Acid : blueprints of life
Replication, Information storage and mutation RIM
– Central Dogma
DNA------->RNA------>protein
Summary 2
• DNA Structure and Function continued:
–
–
–
–
–
Bases are Adenine, Guanine, Cytosine and Thymine- Asian Guys Can Teach: AGCT
Base pairing is A to T and G to C- DNA is where its AT
Sequence of Bases Store information- Like the sequence of numbers in a Phone Number
Sides of the ladder are Sugar-Phosphate backbones
Nucleotides are the building blocks (dNTPs) themselves made of phosphate base and
sugar= PBS- The only station Sierra and Gabriel can watch
– DNA base pairs- DNA velcro (David Letterman
• DNA Replication
– Semi-conservative- Half republican (old) /half democrat (new)
– Template directed with base pairing (AT, GC)
– 5 required ingredients of PCR - primer, template, Mg, dntps, DNA
polymerase (PTMDD)
Summary 3
• Steps in forensic DNA typing are – Evaluation,
Extraction, Quantification, Typing and Interpretation
– 1) Evaluation- Is it there? Screen- blood? Semen?
Saliva, human?
– 2) Extraction- Get and clean DNA
• Open cells -Get DNA
– 3) Quantify- Determine quality and quantity?
• How good and how much did you get?
• Yield Gels, Slot Blots, Real time qPCR assays
Case #2: Can you extract and
type DNA from fingerprint
powder that has been used to
develop latent prints?
If so, design an experiment with
+ and – controls to support your
hypothesis.
RFLP- Rudin and Inman
DNA Methods
1)
2)
3)
Extract
Quantitate
Distinguish
Size
Content
RFLP : Restriction Fragment Length Polymorphisms
PCR: Polymerase Chain Reaction
RFLP methods require large amounts of undegraded DNA and
the process takes 1-2 weeks.
PCR methods require only small amounts of DNA, are useful on
degraded DNA and require much less time (as little as 1-2 days in
some cases).
• The base sequence can exhibit differences in
length and content between individuals.
Christina Aguilera
Lady Gaga
The Suburbs
Lady Antibellum
Esperanza Spalding
Eminem
Train
Norah Jones
Boyz to Men
... AAAGAAAGAAGAAAC...
... AAAGAAAGAAGA...
... AAAGAAAGAAGT...
... AAAGAAAGAAGA...
... AAAGAAAGAA...
... AAAGAAAGA...
... AAAGAAAGC...
... AAAGAAAGT...
... AAAGAAAG…
• Although different between individuals* DNA is identical in
every cell of an individuals body** Some
exceptions*identical twins**diseased individuals, mtDNA
(sport analogs)
Restriction Fragment Length Polymorphisms
(RFLP)
LA
...GGCCAAAGAAAGAAAGGCC...
15
CA
...GGCCAAAGAAACGGCC...
12
SL
...GGCCAAAGAATGGCC...
11
GGTTTCTT : Probe
1) Cut DNA with Restriction Enzymes that
recognize specific sequences : GGCC.
2) Separate the many fragments produced by gel
electrophoresis. The fragments represent a
wide range of sizes.
3) Blot and probe the fragments with specific
DNA sequences that base pair only with
those that contain the sequence of interest.
RFLP
Gel Electrophoresis separates fragments by size
DNA Probe base pairs with specific fragments
M
BIG
Small
LA
CA
SL
M
RFLP Spencer
Comparison of RFLP and PCR
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 813 loci (requires
more loci)
Relative power of tests
•
•
•
•
•
•
•
•
•
Test type
RFLP-VNTR
PCR:
DQAlpha- macroarray
PM - macroarray
D1S80 - gel- VNTR
STRs
-gel,CE, arrays
mtDNA- gel, CE, arrays
alu
-gel, CE, arrays
time
weeks
power
+++ *
1 day
1 day
2 days
2 days
2 days
2 days
+
++
++
+++
+
++
• * not useful on degraded DNA
Summary 1
• Why study DNA
– Law enforcement, evolution, agricultural, and human
applications-medical diagnostics
• DNA Biology and Genetics
– DNA is contained in cells –the basic unit of life
– Found in nuclei, mitochondria and chloroplasts
– Organized in chromosomes. Located at positions called loci and
come in different forms or alleles.
– Homozygous if the same, heterozygous if different
– Alleles segregate independently and assort randomly when on
different chromosomes. Random assortment is desired for forensic
DNA loci.
• DNA Function and Structure
– DeoxyriboNucleic Acid : blueprints of life
Replication, Information storage and mutation RIM
– Central Dogma
DNA------->RNA------>protein
Summary 2
• DNA Structure and Function continued:
– Bases of DNA are Adenine, Guanine, Cytosine and Thymine- Asian Guys Can
Teach: AGCT
– Base pairing is A to T and G to C- DNA is where its AT
– Sequence of Bases Store information- Like the sequence of numbers in a
Phone Number
– Nucleotides are the building blocks (dNTPs) themselves made of phosphate
base and sugar= PBS- The only station Sierra and Gabriel can watch
– DNA base pairs- DNA velcro (David Letterman
• DNA Replication
– Semi-conservative- Half republican (old) /half democrat
(new)
– Template directed with base pairing (AT, GC)
– 5 required ingredients- primer, template, Mg, dntps, DNA
polymerase (PTMDD)
Quantification References
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Anderson S, Howard B, Hobbs GR, Bishop CP. (2005) A method for determining the age of a bloodstain. Forensic Sci Int. 2005 Feb 10;148(1):37-45. Links
Andreasson H, Gyllensten U, Allen M. (2002) Real-time DNA quantification of nuclear and mitochondrial DNA in forensic analysis. Biotechniques. 2002 Aug;33(2):402-4, 407-11.
Andreasson H, Nilsson M, Budowle B, Lundberg H, Allen M. (2006) Nuclear and mitochondrial DNA quantification of various forensic materials. Forensic Sci Int. 2006 Jan 18;
Budowle B, Hudlow WR, Lee SB, Klevan L. (2001) Using a CCD camera imaging system as a recording device to quantify human DNA by slot blot hybridization. Biotechniques. 2001
Mar;30(3):680-5.
Butler, J (2005) Forensic DNA Typing: Biology and Technology Behind STR Markers ISBN: 0-12-147952-8, 688pp. Academic Press.
Fox JC, Cave CA, Schumm JW. (2003) Development, characterization, and validation of a sensitive primate-specific quantification assay for forensic analysis. Biotechniques. 2003
Feb;34(2):314-8, 320, 322. Links
Green RL, Roinestad IC, Boland C, Hennessy LK. (2005) Developmental validation of the quantifiler real-time PCR kits for the quantification of human nuclear DNA samples. J Forensic
Sci. 2005 Jul;50(4):809-25.
Heid CA, Stevens J, Livak KJ, Williams PM. (1996) Real time quantitative PCR. Genome Res. 1996 Oct;6(10):986-94.
Horsman KM, Hickey JA, Cotton RW, Landers JP, Maddox LO. (2006) Development of a human-specific real-time PCR assay for the simultaneous quantitation of total genomic and male
DNA. J Forensic Sci. 2006 Jul;51(4):758-65.
Jones (2005)- Forensic Science Handbook Saferstein editor Forensic Science Handbook, Volume II, 1/e Richard Saferstein, Bill Bliss, Arlington, VA ©1988 / ISBN: 0133268772
Juusola J, Ballantyne J. (2003) Messenger RNA profiling: a prototype method to supplant conventional methods for body fluid identification. Forensic Sci Int. 2003 Aug 12;135(2):85-96.
Juusola J, Ballantyne J. (2005) Multiplex mRNA profiling for the identification of body fluids. Forensic Sci Int. 2005 Aug 11;152(1):1-12.
Kline, M.C., Duewer, D.L., Redman, J.W., Butler, J.M. (2003) NIST mixed stain study 3: DNA quantitation accuracy and its influence on short tandem repeat multiplex signal intensity.
Anal. Chem. 75: 2463-2469.
Kline, M.C., Duewer, D.L., Redman, J.W., Butler, J.M. (2005) Results from the NIST 2004 DNA Quantitation Study. J. Forensic Sci., 50(3): 571-578.
Mandrekar MN, Erickson AM, Kopp K, Krenke BE, Mandrekar PV, Nelson R, Peterson K, Shultz J, Tereba A, Westphal N. (2001) Development of a human DNA quantitation system.
Croat Med J. 2001 Jun;42(3):336-9.
Nicklas JA, Buel E. (2003) Development of an Alu-based, QSY 7-labeled primer PCR method for quantitation of human DNA in forensic samples, Journal of Forensic Sciences, Vol 48,
No 2, 282-291, 2003.
Nicklas JA, Buel E, (2003) Development of an Alu-based, real-time PCR method for quantitation of human DNA in forensic samples, Journal of Forensic Science, Vol 48, No 5, 936-944,
2003.
Nicklas JA, Buel E, (2003) Quantitation of DNA in Forensic Samples, Analytical and Bioanalytical Chemistry, Vol 376, No. 8, 1160-1167, 2003.
Nicklas JA, Buel E. (2006) Simultaneous determination of total human and male DNA using a duplex real-time PCR assay. J Forensic Sci. 2006 Sep;51(5):1005-15
Nussbaumer C, Gharehbaghi-Schnell E, Korschineck I. (2006) Messenger RNA profiling: a novel method for body fluid identification by real-time PCR. Forensic Sci Int. 2006 Mar
10;157(2-3):181-6. Epub 2005 Nov 9.
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Steps in Forensic DNA typing
Evaluation- Is it there?
(Figure 6.1 Rudin and Inman 2001)
1.
Start with biological sample
2.
Screen- blood? Semen? Saliva, human?
Extraction- Get and clean DNA
3.
Open cells  Get DNA
4.
Methods to get DNA and purify DNA
Quantify- Determine quality and quantity?
5.
QuantifyHow good and how much did you get?
Type to determine and compare alleles
6. RFLP vs PCR
7. Determine alleles and compare DNA types
Or alleles present in samples and references
Interpretation of Results
DNA Methods
1)
2)
3)
Extract
Quantitate
Distinguish
Size
Content
RFLP : Restriction Fragment
Length Polymorphisms
PCR: Polymerase Chain Reaction
RFLP methods require large amounts of undegraded DNA and
the process takes 1-2 weeks.
PCR methods require only small amounts of DNA, are useful on
degraded DNA and require much less time (as little as 1-2 days in
some cases).
RFLP Spencer
Classroom RFLP
• Everyone get a base- Blue=A, Green=G,
Yellow=C, Red =T
• Form GGCCAATTCCGGTTAAGATC- hold
hands or papers (It is valentine’s day!)
• You are the DNA we will cut and separate on a gel
(in the class)
• Set DNA in the ‘well’= front of class
• Enzyme role= Cut between GG-CC
• Turn on power (electrophoresis). Migrate through
the gel (classroom desks).
• Stop when power is off. Observe separation
Kary Mullis Nobel Prize - 1993
Polymerase Chain Reaction:
PCR is simply repeated rounds of DNA replication
PCR based systems are rapid,
require less material than RFLP
and less time for typing
• Molecular xeroxing
• Calvin and Hobbes example
PCR works for very small
samples—bloodstain on hat
PCR works for very small
samples—hat close-up
PCR works for degraded DNA—under
the microscope,
sperm appear intact
But the yield gel shows that
their DNA is degraded
PCR: repeated rounds of DNA
Replication
5-P’
•5 required ingredients (components)- primer,
template, Mg, dntps, DNA polymerasePTMDD-(please to make DNA doubled)
Primer
5’
HO
P
O-
3-OH’
O
•DNA Polymerase catalyzes the template
directed (A-T, G-C), incorporation of dNTPs
(PP is released) forming a 3’-5’
phosphodiester linkage
Base
(A, T, C, or G)
O
CH 2
O
H
H
H 3’
O
2’
4’
• Direction of synthesis 5’3’ using primer
3’OH to attach incoming nucleotide
1’
H
Base
(A, T, C, or G)
H
5’
HO
P
O-
O
CH 2
O
4’
H
1’
H
H 3’
2’
OH H
3-OH’
P-P-HO
Mg++
Template = Old
dNTP Base
O
H
DNA polymerase
P
O-
O
CH 2
O
H
4’
1’
H
H 3’
2’
OH H
O
H
5’
HO
5-P’
(A, T, C, or G)
5’
P
O-
O
CH 2
O
DNA Amplification with the
Polymerase Chain Reaction (PCR)
5’
3’
5’
3’
3’
3’
5’
5’
Starting DNA
Template
Separate
strands
(denature)
Forward primer
5’
3’
5’
3’
Make copies
Add
primers
(extend
primers)
5’
(anneal)
3’
3’
5’
Reverse primer
In 32 cycles at 100% efficiency, 1.07 billion
copies of targeted DNA region are created
PCR takes place in a Thermal
Cycler
Thermal Cycling Temperatures
Temperature
94 oC
72 oC
60 oC
94 oC
94 oC
94 oC
72 oC
60 oC
72 oC
60 oC
Single Cycle
Time
The denaturation time in the first cycle is
lengthened to ~10 minutes when using
AmpliTaq Gold to perform a “hot-start” PCR
Typically 25-35 cycles
performed during PCR
PCR Process
5’
3’
Starting DNA
Template
3’
5’
Separate
strands
(denature)
Forward primer
5’
3’
3’
5’
5’
Add primers
(anneal)
3’
3’
5’
Reverse primer
Make copies
(extend primers)
Repeat Cycle,
Copying DNA
Exponentially
PCR is simply repeated rounds of DNA
replication
Template- DNA from blood etc.
5’
3’
3’
Step 1: Denature
Separate H bonds with heat at 95C
5’
95C
5’
3’
3’
5’
55C
Step 2: Anneal
Primers bind at lower temp 55C
5’
3’
5’
3’
5’
3’
5’
3’
72C
Step 3: Extend
Taq polymerase extends primer 3’OH 5’
at 72C (dNTPs and Mg++)
3’
Step 4: Repeated 28-30 rounds of D, A,
E
3’
5’
Number of Target Molecules
Created
Cycle Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Number of Double-stranded Target Molecules
0
0
2
4
8
16
32
64
128
256
512
1024
2048
4096
8192
16,384
32,768
65,536
131,072
262,144
524,288
1,048,576
2,097,152
4,194,304
8,388,608
16,777,216
33,544,432
67,108,864
134,217,728
268,435,456
536,870,912
1,073,741,824
Comparison of RFLP and PCR
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 813 loci (requires
more loci)
Relative power of tests
•
•
•
•
•
•
•
•
•
Test type
RFLP-VNTR
PCR:
DQAlpha- macroarray
PM - macroarray
D1S80 - gel- VNTR
STRs
-gel,CE, arrays
mtDNA- gel, CE, arrays
alu
-gel, CE, arrays
time
weeks
power
+++ *
1 day
1 day
2 days
2 days
2 days
2 days
+
++
++
+++
+
++
• * not useful on degraded DNA
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.
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
Multiplex PCR
• Target 2 or more DNA regions simultaneously
with multiple primer sets. Copy more than one
locus at a time
• Primers for all loci are present in the tube
• Conditions are adjusted to ensure all loci will be
amplified
• Multiple types obtained from 1-2 ng DNA
• Greater discrimination
• Advantages:
– more information in the same amount of time
– less expensive (lower reagents and labor)
• Challenge lies in designing PCR primers that are
compatible with one another
Primer Design
• Typically performed with assistance of computer
program to identify possible primer that are then
tested empirically
• Various computer programs:
– Gene Runner (PC), Oligo (PC/Mac), Primer Express (Mac)
– Primer 3 (web based)
• Critical parameters examined:
–
–
–
–
Predicted Tm (melting temperature)- Tm=4(G+C) + 2(A+T)
Primer dimer and hairpin formation
Contiguous base runs (usually <5 bases)
GC content (number of G and C nucleotides within primer)
Schematic of Multiplex PCR
Locus A
Locus
C
Locus
B
A
B
C
small
large
Multiplex PCR
• Over 15 Markers Can Be
Copied at Once
• Sensitivities to levels less than
1 ng of DNA
• Ability to Handle Mixtures
and Degraded Samples
• Different Fluorescent Dyes
Used to Distinguish STR
Alleles with Overlapping Size
Ranges
Important PCR facts
• DNA polymerase is taq polymerase from a hot springs
(can survive denaturation boiling temperatures)
• Taq likes to add an extra base (non template directed
nucleotide addition to the 3’ end). Amplification of DNA
fragments of 100bp in size are 101 in length.
• PCR amplification sometimes “stutters” on STRs
resulting in an extra PCR product called a stutter product.
Eg. Both 100 (correct type) and 96 base pair fragments
are present. The stutter product is usually represented at
less than 10% of the real allele.
Real Time PCR
Quantitative PCR- QPCR
http://pathmicro.med.sc.edu/RTPCR/rt-pcr.ppt
• Real-time QPCR has several advantages over the other methods in
that it is extremely accurate and sensitive over a broad dynamic
range, and it occurs in a closed-tube system, reducing the potential
for carryover contamination.
• Using this technique, a forensic biologist can monitor and quantify
the accumulation of PCR products during log phase amplification.
(Heid et al., 1996).
• Several RT PCR human specific assays are now available that target
autosomal, Alu repeats, Y chromosome and mtDNA (Andréasson et
al. 2002, Nicklas et al. 2003, Green et al. 2005, Andréasson et al.
2006, Horsman et al. 2006).
• The assays may be performed on single targets or in multiplexes
(Timken et al. 2005, Walker et al. 2005, Nicklas et al. 2006).
• Recently, the detection of degraded vs intact human DNA and PCR
inhibitors has been reported (Swango et al. 2006).
RNA based quantification methods
• Different genetic expression patterns (mRNAs) exist in different tissue
types.
• Body fluid identification has been reported based on their mRNA
profiles (Juusola and Ballantyne 2003 and 2005, Nussbaumer et al.
2006)
• In addition, the age of a bloodstain was reported using analysis of
mRNA: rRNA ratios (Anderson et al. 2005). This information may be
useful in establishing the time of the crime.
• Advantages of the mRNA-based approach, versus the conventional
biochemical tests, include greater specificity, simultaneous and semiautomatic analysis, rapid detection, decreased sample consumption
and compatibility with DNA extraction methodologies.
• The quantification of the amounts of the mRNA species relative to
housekeeping genes is a critical aspect of the assays (Juusola and
Ballantyne 2003).
Comparison of Methods used for
DNA Quantification
Method
UV Spectrophotometry
Yield Gel electrophoresis
Slot Blot
Yield Gel blot
Pico-green microtitre plate
Ease
+++++
+++
++
+
++++
Cost
+
+
++
++
++
Sensitivity
++
+
+++
+++
++++
Result
Total DNA
Int vs deg DNA
Human DNA
Int. vs. deg human DNA
Total DNA
Alu Quant
Real time PCR assays
Real time PCR assays
+++
+++
+++
+++
+++
+++
++++
+++++
+++++
Human DNA
Human DNA
Int.vs. deg.Human DNA
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.
Do not move from PCR area into non PCR area without decontamination
Process one sample at a time, Avoid splashing
Separate reference samples from evidence
Wear protective gear and reagent prep care
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
Controls: Negative, Positive, Stochastic, Substrate
Monitoring for Contamination—
Controls ‘R’ Us
Bloodstain (Evidence)
Substrate Control
Reagent Blank—for Evidence
Victim’s Reference Sample
Reagent Blank—for References
Negative Amplification Control
Quality Control Sample
Positive Amplification Control
PCR ‘quiz’
• Template =
• 5’GGACTCCTATGTATGTATGCTTTAAGGCA 3’
3’CCTGAGGATACATACATACGAAATTCCGT 5’
• Design two primers (five bases long): Remember-the 3’
OH end will be extended and DNA is antiparallel
• Be sure to amplify the entire template.
• List the other required components, materials and
procedure needed to conduct a successful PCR reaction
• Assume this is an STR locus. What is the repeat unit?
What is the type (number of repeats for this allele)?
Reverse Dot blot hybridization
eg. DQ alpha and Polymarker
Once amplified detection can be done by DNA battleship
DNA probes can detect specific fragments by
base pairing (complementation:hybridization)
1
2
PROBE
TCT TTC
JB
AAAGAAAGCCAG
JM
AAAGAAGGCC
DNA probes can detect specific fragments by
base pairing
MW (complementation:hybridization)
AAAGAAACCC
1
2
LGPROBE
AAAGAACCC
TTC
TCT
JB
AAAGAAAGCCAG
SL JM AAAGAATT
AAAGAAGGCC
MW AAAGAAACCC
Plant
LG AAATTCCC
AAAGAACCC
1
SL
AAAGAATT
Plant AAATTCCC
2
1
TCT
TCT
2
TTC
TTC
TCT
TCT
TTC
TTC
TCC
TGG
TAA
TCC TGG
TCC
TCC
TGG
TGG
Plant
DNA
TAA
TAA
SL
DNA
TAA
JB
DNA
Plant
DNA
SL
Summary
• RFLP (old) required approximately 50ng of DNA
at a minimum. PCR requires as little at 500pg or
100 times less!
• One method to examine variation of variable
number of tandem repeats (VNTRs) is RFLP=
restriction fragment length polymorphisms
• RFLP requires many steps, undegraded DNA and
takes days to weeks to complete
• In contrast, typing of STRs using PCR can be
performed on very small amounts of degraded
DNA and takes hours to a day to complete.
Summary 2
• PCR is polymerase chain reaction and is repeated
rounds of DNA synthesis.
• There are 5 components needed, PTMDD.
• PCR takes place in a thermal cycler
• Multiplex PCR permits amplification of many loci
simultaneously and saves time
• Avoid contamination and use controls
• Other markers that have been used in forensic PCR
assays include, dot blot assays of DQ alpha,
polymarker, and D1S80.
• Mitochondrial DNA sequencing and Y chromosome
STR markers are also being used.