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DNA = Deoxyribonucleic Acid

Located in CHROMOSOMES in the nucleus of cells ◦ What is a chromosome?

Tightly packed genetic information

◦ Where do we get them from?

One from each parent!

Genes – portions of DNA that code for traits and functions

35,000 genes in human body

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DNA

Timeline

1868 Miescher “discovers” DNA 1953 Watson and Crick report double helix structure 1977 First human gene cloned. 1984 Jeffreys reports DNA sequences 1985 First report of PCR method 1986 Jeffreys uses DNA to solve first murder case 1987 First conviction on DNA evidence 1991 STRs first reported 1998 FBI starts CODIS database

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D Si r Al ec

• Discovered in 1984

by Dr. Alec Jeffreys

at the University of

Leicester

• He was knighted for

his discovery

Discovery

Case Study: The First Use of DNA Evidence

• Two teenage girls raped and murdered in Leicestershire, England

• Semen from the victims indicated a male with Type A blood and a rare enzyme (10% of the local male population)

• A local boy, Richard Buckland, confesses upon interrogation

• Police use DNA fingerprinting to confirm, but DNA profiles of Buckland and crime scene DNA do not match

• Ironically, Buckland becomes the first person exonerated by DNA evidence

Case St udy: The Fi rst Use of DNA Evi dence

• Police request DNA samples from all adult males in 3 nearby villages (5000 men)

• 6 months later – no results!

• A year later, police are informed by a bakery worker that they overheard a co-worker bragging they had given a DNA sample for another man

• Police obtain DNA from Colin Pitchfork and obtain a perfect match

• In 1988, Colin Pitchfork was tried and convicted and sentenced to life in prison for the double rape and homicide based in large part to the DNA evidence

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DNA is a POLYMER made up of ◦ What is a Polymer?

A large molecule made up of repeating structures

Nucleotides are made up of three parts ◦ Sugar (deoxyribose)

◦ Phosphate

◦ A nitrogen containing base (A,T,G,C)

There are approximately 100 million nucleotides in the average DNA molecule!

A, G, C or T

What makes DNA Different from RNA?

Forms sugar Phosphate Backbone

◦ Adenine (A)

◦ Guanine (G)

◦ Thymine (T)

◦ Cytosine (C)

Base pairing

Bases bind with SPECIFICITY!! ◦ A-T

◦ G-C

The order of the bases is what distinguishes

different DNA strands.

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Nucleic Acid Polymer (DNA)

Phosphate

Sugar

Base

Phosphate

Sugar

Phosphate

Sugar

Phosphate

Base

Base Polymer

Can predict sequence of one strand based on the sequence of the other.

Responsible for Replication and Transcription

. Example: GAG codes for the amino acid glutamine, while CGT

codes for alanine ◦ If a nucleotide is changed the “wrong” amino acid is placed in the

protein and the protein may not function correctly and this is the basis for many diseases and health issues.

The specific sequence of nucleotides of all human beings is 99.9% the SAME!!

It is that 0.1% difference that makes each person unique ◦ What is the exception to this rule?

Identical twins

◦ What is so important about the sequence?

It is a code for amino acids proteins

A T C G A C T A A C C G A C

T A G C T G A T T G G C T G

Uses of DNA fingerprinting ◦ Forensic cases – matching suspects with evidence

◦ Clear persons wrongfully accused

◦ ID crime and catastrophe victims

◦ Establish paternity and other familial relations

◦ Match organ donors with recipients in transplant programs

◦ Missing persons investigations

Restriction Fragment Length Polymorphism ◦ Characterize fragments and calculate the statistical

probability that two people could have the same fragment sequence

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Portions of DNA contain sequences of bases that are repeated numerous times, known as

These tandem repeats offer a means of distinguishing one individual from another.

They act as filler or spacers between the coding regions of DNA.

What is important to understand is that all humans have the same type of repeats, but there is tremendous variation in the number of repeats each of us have.

cut DNA into fragments. They are thought of as highly specialized scissors that cut a DNA molecule when it recognizes a specific sequence of bases.

Length differences associated with relatively long repeating DNA strands are called

and form the basis for one of the first DNA typing procedures.

Typically, a core sequence consists of 15 to 35 bases in length and repeats itself up to a thousand times.

The key to understanding DNA fingerprinting lies in the knowledge that numerous possibilities exist for the number of times a particular sequence of base letters can repeat itself on a DNA strand.

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DNA “Fingerprinting” RFLP Restriction enzyme cuts at ….GAATTC…..

Spacing between cutting sites for enzyme is different for different individuals

Once the DNA molecules have been cut up by a restriction enzyme, the resulting fragments are sorted out by

The smaller DNA fragments will move at a faster rate on the gel plate than the larger ones.

The fragments are then transferred to a nylon membrane

To visualize the RFLPs, the nylon sheet is treated with radioactive probes containing a base sequence complementary to the RFLPs being identified

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RFLP: El ect rophoresi s • DNA is visualized using electrophoresis

• Negatively charged DNA moves through a gel with a current

• Smaller DNA moves faster than larger DNA fragments

Which Suspect, A

or B, cannot be

excluded from the

class of potential

perpetrators of

this assault?

Need a relatively large amount of DNA

Large sequence size/lengths (do not allow for copying)

Requires a good sample (undegraded)

Relatively slow

What to do when there’s not much there…

PCR can amplify minute quantities of DNA many

millions of times.

Uses high temperature enzyme that “mindlessly”

copy DNA between “start” and “stop” markers

(primers).

After just 20 cycles a single copy of DNA is amplified

over 2,000,000 fold.

Can be automated

Can use very small samples!

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PCR - DNA “Fingerprinting”

Steps:

Isolate DNA piece

1. Heat/Denature

separation of DNA Strands

2. Annealing use primers [“start” and “stop” switches] to anneal

[bind to complimentary DNA sequences]

3. Extension

use polymerases to build complimentary strand

between “switches”

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DNA Amplification - PCR

Separated into 2 strands (Denaturation) ◦ Heat or chemically

G

T

A

G A

A T

C

A

T

C

T

- attaching primers (“start” and “stop” switches for DNA replication.

- using polymerases

T

T

Completes first cycle - cycles repeated many times.

Primer - fixed DNA binding

points.

PCR technology cannot be applied to RFLP since the strands are too long (often numbering in the thousands of bases)

PCR is best used with DNA strands that are no longer than a couple of hundred bases.

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The latest method of DNA typing uses analysis.

STRs are locations on the chromosome that contain short sequences that repeat themselves within the DNA molecule.

Serve as useful markers for identification since they are found in great abundance throughout the human genome

Normally consist of repeating sequences of 3 to 7 bases in length

Because they are short, STRs are ideal candidates for multiplication by PCR

STRs are much less susceptible to degradation and may often be recovered from stains that have been subjected to decomposition

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Hundreds of different types of STRs are found in human genes.

The more STRs one can characterize, the smaller the percentage of the population from which the sample can come from

Gives rise to the concept of

Using PCR, simultaneous extraction and amplifcation of a combination of different STRs can be done (over 10 markers can be copied at once)

Shorter DNA strands would be expected to be more stable and less subject to degradation

Offers the advantage in that it can amplify minute quantities of DNA, thus overcoming the limited sample size (100 times less than that normally required for RFLP analysis)

Works well on fragmented DNA

Rapid analysis

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Another tool available for crime scene investigations are STRs located on the Y chromosome, which is male specific.

More than 20 different Y-STR markers have been identified

Y-STRs will prove useful when multiple males are involved in a sexual assault or in question

The Y-STR is less complicated in appearance and interpretation.

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Nuclear DNA ◦ Located in nucleus.

◦ Inherited 1/2 from mother and 1/2 from father.

Mitochondrial DNA ◦ Located in mitochondria

◦ Inherited solely from mother.

Where is DNA

Cellular Matter ◦ Organs

◦ Hair Roots (nuclear)

◦ Hair Shaft (mitochondrial)

◦ Sweat

◦ Semen

◦ Blood

◦ Saliva

◦ Sw eat

◦ Bone

◦ Fingernail Scraping

Mitochondrial DNA

Nuclear DNA

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Shows maternal lineage matches (will be identical if related through the mother)

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Used when samples cannot be analyzed by RFLP or STR.

Older biological samples that lack nucleated cell material, such as

hair, bones, and teeth, can be analyzed with mtDNA

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FBI’s CODIS DNA Database

Combined DNA Index System Used for linking serial crimes and unsolved cases with repeat

offenders

Launched October 1998

Requires markers

The Forensic Index contains DNA profiles from crime scene evidence.

The Offender Index contains DNA profiles of individuals convicted of sex offenses (and other violent crimes) with many states now expanding legislation to include other felonies

◦ Forensic Profiles in NDIS (National DNA Index system – a component of CODIS): 119,782

◦ Convicted Offender Profiles in NDIS: 2,643,409

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A Sampl e Pr of i l e

• By combining the frequency information for all 13 CODIS loci, the frequency of this profile would be 1 in 7.7 quadrillion Caucasians