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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

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Where do we get the DNA?

• Blood

• Semen

• Saliva

• Urine

• Hair

• Teeth

• Bone

• Tissue

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DNA in the Cell

chromosome

cell nucleus

Double stranded

DNA molecule

Individual

nucleotides

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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

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Review of Replication

Two polymers separate

Each serves as a template

DNA polymerase adds nucleotides,

but not without a primer…

DNA: Nucleotide structure

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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

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DNA Polymerase requires a primer

WHY?

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DNA Polymerase requires the primer’s 3’OH to add the next nucleotide

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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

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Assessment 1. Name four

molecules required to make new

DNA.

• Template DNA

• Primers

• Nucleotides (A, T, G, C)

• DNA Polymerase

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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

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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

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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

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polymers

Nucleotides

complementary

ladder

steps

DNA polymerase

primers

PCR template

Assessment 2.

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STRs are Polymorphisms • Small Tandem Repeats

• Polymorphism: two or more distinct forms of a section of DNA

7 repeats

8 repeats

AATT

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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

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The STRs used for DNA

Fingerprinting

CSF1PO

D5S818

D21S11

TH01

TPOX

D13S317

D7S820

D16S539 D18S51

D8S1179

D3S1358

FGA

VWA

AMEL

AMEL

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STRs can be replicated in

a test tube.

Target Region for

Replication

chromosome

cell nucleus

Double stranded

DNA molecule

Individual

nucleotides

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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

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Forensics labs use

Multiplex PCR –

many STRs

copied at once

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DNA has a negative charge. If put into an electric

field, DNA will migrate towards the positive

electrode.

ROPE TRICK

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Viewing the DNA

• Stain with

ethidium bromide

– fluoresces in UV

light

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Analysis of a Single DNA

Polymorphism

• PCR results of

amplification of the

D1S80

– Range of fragment

sizes = 224 to 640 bp

400 bp

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Looking at

many

polymorphisms

at once

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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)

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