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1
DNA FINGERPRINTING MADE
EASY FOR FORENSICS Presented by
Eilene Lyons
The St. Louis Community College – Florissant Valley
Biotechnology Program
Some slides are from a downloaded PPT presentation from The National Institute of Standards and Technology (NIST)
http://www.cstl.nist.gov/biotech/strbase/training.htm
2
Where do we get the DNA?
• Blood
• Semen
• Saliva
• Urine
• Hair
• Teeth
• Bone
• Tissue
3
DNA in the Cell
chromosome
cell nucleus
Double stranded
DNA molecule
Individual
nucleotides
4
5
REVIEW OF DNA STRUCTURE
• NUCLEOTIDE monomers – A, T, G, C
• DNA POLYMER - long chain of nucleotides
• HYRDROGEN BONDS hold two polymers
together to form a ladder
• COMPLEMENTARY nucleotides make the
steps of the ladder
– A bonds with T
– G bonds with C
6
Review of Replication
Two polymers separate
Each serves as a template
DNA polymerase adds nucleotides,
but not without a primer…
DNA: Nucleotide structure
7
A nucleotide is composed of a nucleobase (nitrogenous base),
a five-carbon sugar (either ribose or 2'-deoxyribose), and one to
three phosphate groups. Together, the nucleobase and sugar
comprise a nucleoside. The phosphate groups form bonds with
either the 2, 3, or 5-carbon of the sugar, with the 5-carbon site
most common. Cyclic nucleotides form when the phosphate
group is bound to two of the sugar's hydroxyl groups.[1]
Ribonucleotides are nucleotides where the sugar is ribose, and
deoxyribonucleotides contain the sugar deoxyribose.
Nucleotides can contain either a purine or pyrimidine base.
Nucleic acids are polymeric macromolecules made from
nucleotide monomers. In DNA, the purine bases are adenine
and guanine, while the pyrimidines are thymine and cytosine.
RNA uses uracil in place of thymine.
Ribose structure indicating numbering of carbon atoms
8
DNA Polymerase requires a primer
WHY?
9
DNA Polymerase requires the primer’s 3’OH to add the next nucleotide
10
11
Review of DNA Replication
• POLYMERS SEPARATE - Hydrogen bonds break to unzip the sides of the ladder
• TEMPLATE – one polymer of the DNA
• PRIMERS – complementary RNA added to the template
• NUCLEOTIDES – the monomers (A, T, G, C)
• DNA POLYMERASE – the enzyme that adds new complementary nucleotides starting at the 3’ OH end of the RNA primer
13
Assessment 1. Name four
molecules required to make new
DNA.
• Template DNA
• Primers
• Nucleotides (A, T, G, C)
• DNA Polymerase
14
STEP 1:
DENATURATION
HEATING TO 94C
STEP 2: PRIMER
ANNEALING
primer
primer
COOLING TO 58C
STEP 3: EXTENSION HEATING TO 72C
FIGURE 3. Steps of PCR
primer
primer
Polymerase Chain Reaction: DNA Replication in a test tube
15
Starting DNA
Template
5’
5’
3’
3’
DNA Amplification with the
Polymerase Chain Reaction (PCR)
Separate
strands
(denature)
5’
5’ 3’
3’
Make copies
(extend primers) 5’
5’
3’
3’
Add primers
(anneal)
5’ 3’
3’ 5’
Forward primer
Reverse primer
16
In 32 cycles at 100% efficiency, 1.07 billion
copies of targeted DNA region are created
PCR Copies DNA Exponentially
through Multiple Thermal Cycles
Original DNA target region
Thermal cycle Thermal cycle Thermal cycle
17
polymers
Nucleotides
complementary
ladder
steps
DNA polymerase
primers
PCR template
Assessment 2.
18
STRs are Polymorphisms • Small Tandem Repeats
• Polymorphism: two or more distinct forms of a section of DNA
7 repeats
8 repeats
AATT
19
People are Diploid
• We inherit
polymorphisms from
our parents.
• Everyone has 2
copies of each piece
of DNA:
– One chromosome
from Mom
– One chromosome
from Dad
20
The STRs used for DNA
Fingerprinting
CSF1PO
D5S818
D21S11
TH01
TPOX
D13S317
D7S820
D16S539 D18S51
D8S1179
D3S1358
FGA
VWA
AMEL
AMEL
21
STRs can be replicated in
a test tube.
Target Region for
Replication
chromosome
cell nucleus
Double stranded
DNA molecule
Individual
nucleotides
22
PCR Replicates STRs
Polymerase Chain
Reaction
What must be added to
the tube?
1. Template DNA
2. Primers
3. DNA Polymerase (Taq from Thermus
aquaticus)
4. Nucleotides
23
Forensics labs use
Multiplex PCR –
many STRs
copied at once
24
DNA has a negative charge. If put into an electric
field, DNA will migrate towards the positive
electrode.
ROPE TRICK
26
Viewing the DNA
• Stain with
ethidium bromide
– fluoresces in UV
light
27
Analysis of a Single DNA
Polymorphism
• PCR results of
amplification of the
D1S80
– Range of fragment
sizes = 224 to 640 bp
400 bp
28
Looking at
many
polymorphisms
at once
29
Check out these sites:
• http://www.dnalc.org/ddnalc/resources/pcr.
html
• http://www.fbi.gov/hq/lab/codis/index1.htm
• http://www.cstl.nist.gov/biotech/strbase/ind
ex.htm
DNA replication Diagram (From Wikipedia, not part of EL presentation)
30