DNA Analysis

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

DNA

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DNA Timeline

1868 Miescher “discovers” DNA1953 Watson and Crick report

double helix structure1977 First human gene cloned.1984 Jeffreys reports DNA

sequences1985 First report of PCR method1986 Jeffreys uses DNA to solve

first murder case1987 First conviction on DNA

evidence1991 STRs first reported1998 FBI starts CODIS database

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• 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

• 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

DNA is a POLYMER made up of nucleotides

What is DNA?

Nucleotides are made up of three partsSugar (deoxyribose)PhosphateA nitrogen containing base (A,T,G,C)

There are approximately 100 million nucleotides in the average DNA molecule

Double helix—two coiled DNA strands Composed of nucleotides connected together

Four bases make the rungs: Adenine Cytosine Guanine Thymine

Bases always bind with specificity (complementary base pairing)

A - T G – C

General Structure of DNA

Nucleotide

A, G, C or T

What makes DNA Different from RNA?

Forms sugar Phosphate Backbone

Nucleic Acid Polymer (DNA)

Phosphate

Sugar

Base

Phosphate

Sugar

Phosphate

Sugar

Phosphate

Base

BasePolymer

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

Responsible for Replication and Transcription◦Every three nucleotides in DNA codes for one amino acid in the formation of a protein.

◦ 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.

What is important about base pairs?

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 the coding for proteins

Order in the Court!

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

Match the sequence!

Forensic cases – matching suspects with evidence

Exonerate individuals ID crime and catastrophe victims (ex. 9/11) Establish paternity and other familial relations Match organ donors with recipients in transplant

programs Missing persons investigations

Forensic Use of DNA

Bodily sources of DNA

All Cells that have a nucleus: white blood cells (NOT RED BLOOD

Cells-they don’t have a nucleus) Semen saliva –contains buccal (cheek

cells) hair root Teeth bone Any tissue

All cells contains thousands of mitochondria (which contain maternal DNA)

DNA “Typing” (Fingerprinting/Analysis)

3 main technologies have been used:

1) RFLP –Restriction Fragment Length Polymorphisms - Developed in 1985 & used until mid 1990’s

2) PCR –Polymerase Chain Reaction- Developed in the 1980’s, but perfected in mid

1990’s- Still used today as a step in the STR process

3) STR-Short Tandem Repeats- Developed in the 1990’s - Used almost exclusively today

Restriction Fragment Length Polymorphism◦ Characterize fragments and calculate the

statistical probability that two people could have the same fragment sequence

The RFLP Technique

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

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.

Restriction enzymes 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 restriction fragment length polymorphisms (RFLP)

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

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General Overview:

1)Isolate—separate DNA from the cell of desired individuals & evidence

2)Cut—using restriction enzymes to cut DNA into smaller fragments

3)Separate/ Sort—by size using electrophoresis which creates banding pattern.

4)Analyze—the specific allele patterns for identification

RFLP technique

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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 electrophoresis.

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

The nylon sheet is treated with radioactive probes to visualize the RFLPs

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Analysis

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RFLP: Electrophoresis• 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?

Three Possible Outcomes Match —The DNA profile appears the same.

Excluded—The two samples originate from different sources.

Inconclusive—The data does not support a

conclusion as to whether the profiles match.

Need a relatively large amount of DNA Large sequence size/lengths (do not allow for

copying) Requires a good sample (undegraded) Relatively slow Crime scene DNA is often in adverse

conditions

Down-side of RFLP

What to do when there’s not much there…

Technique used for making copies of DNA molecule

PCR can amplify minute quantities of DNA many millions of times.

Uses high temperature enzyme that “mindlessly” copy DNA

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 (Polymerase Chain Reaction)

PCR process - DNA “Fingerprinting”

Steps:Isolate DNA piece1. Heat/Denature: separation of DNA Strands

(unzip)

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

TG C

ATTA

G CA T

AG

TA

GA

A TC

AT

CT

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

Extending - using polymerases

TAC TA TTCTT AT C

AA TA G G

TAA ATA G G

T ACTT AT C TAC TT

C A

AA

G

GG

GTT T

TAG

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)

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PCR and RFLP

Typically 28-32 PCR cycles are run resulting in billions of copies of DNA

Each cycle that doubles the DNA takes about 2 minutes.

Minute amounts of DNA may be used for amplification (less than 1 billionth of a gram).

Can get enough DNA from envelopes, stamps, soda cans, & cigarette butts to run PCR

Advantages of PCR

The latest method of DNA typing uses short tandem repeat (STR) analysis.

STRs are locations on the chromosome that contain short sequences of 3-7 bases that repeat themselves within the DNA molecule (100-200x).

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

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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Short Tandem Repeats (STRs)

By continuing the process with additional STRs from other genes, one can narrow down the probability of DNA belonging to a person.

STR typing is visualized by peaks shown on a graph. Each represents the size of the DNA fragment.

The possible alleles are numbered for each loci.

Norma's genotype is 15 repeats, 15 repeats at the locus D3S1358, 14 repeats, 16 repeats at vWA, 24 repeats, 25 repeats at FGA (gets on from each parent)

Short Tandem Repeats (STRs)

Using STR DNA Profiling ResultsDavid & Karen are parents of a missing child:

DNA Profile from remains found in a shallow grave:

Could this be their child?

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.

Y- STR

Shorter DNA strands tend to be more stable and less susceptible to degradation

It can amplify minute quantities of DNA- need only 18 DNA bearing cells (100 times less than that normally required for RFLP analysis)

Works well on fragmented DNA

More rapid analysis

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STR-PCR Advantages

Probability of a person matching a random DNA sample at any 1 STRS site is roughly 1/10

3 STRS sites? 1/10 x 1/10 x 1/10 = 1/1000

All 13 STRS sites would mean that the chances of matching a random DNA sample are about 1 in 10 trillion:

1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x = 1/10,000,000,000,000

Probability of two different people matching at all 13 STRS sites is virtually zero.

DNA Identification Based on Probability

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A Sample Profile

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

Where is DNA found?

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Nuclear DNA ◦ Located in the nucleus of cells◦ Inherited ½ from mother and ½ from father

Mitochondrial DNA ◦ Located in mitochondria (found in cytoplasm)◦ Inherited solely from mother◦ Used when nDNA typing is not possible (old,

degraded samples)◦ More costly and time consuming

Nuclear vs. Mitochondrial DNA

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

Mitochondrial DNA

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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 the 13 core STR 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