“The capacity to blunder slightly is the real marvel of DNA. Without this special attribute, we...

“The capacity toblunder slightly is thereal marvel of DNA.Without this specialattribute, we would stillbe anaerobic bacteria andthere would be no music.”

—Lewis Thomas, Physician, author

Chapter 11 Kendall/Hunt Publishing Company 2

DNA Analysis

Students will learn: That DNA is a long-chain polymer found in nucleated cells, which contains genetic information. That DNA can be used to identify or clear potential suspects in crimes. How DNA is extracted and characterized. How to apply the concepts of PCR and STRs to characterize DNA. The role that statistics plays in determining the probability that two people would share a DNA profile


DNA Analysis

Students will be able to: Explain that DNA is a long molecule, tightly

packed in the form of a chromosome containing genetic material.

Isolate and extract DNA from cells. Describe the PCR process Describe STR testing and calculate probabilities

of identity using STR.


Historical Information James Watson and Francis Crick—1953

discovered the configuration of the DNA molecule

Ray White—1980 describes first polymorphic RFLP marker

Alec Jeffreys—1985 isolated DNA markers and called them DNA fingerprints

Kary Mullis—1985 developed PCR testing

1988—FBI starts DNA casework

1991—first STR paper

1994—FBI launches CODIS database


People of Historical Significance

James Watson, Francis Crick, and Maurice Wilkins jointly received the Nobel Prize in 1962 for their determination of the structure of DNA. What is interesting about this fact is that Rosalind Franklin had as much to do with the discovery as the other three men with her work with X-ray crystallography. She died of cancer and could not be honored for her work. Find out more at Chemical Achievers:


Where Is DNA Found? Genes are portions of DNA that code for

specific proteins DNA is found in all nucleated body cells

—white blood cells, semen, saliva, urine, hair root, teeth, bone, tissue

Abundant in cheek cells Red blood cells have no nuclei; and

therefore, no nuclear DNA DNA obtained from blood comes from

white blood cells


General DNA Information Double helix—two coiled DNA strands Composed of nucleotides—units containing a sugar

molecule (deoxyribose), phosphate group and a nitrogen-containing base

In humans, the order of these bases is 99.9% the same.

Four bases Adenine Cytosine Guanine Thymine

Bases always pair A to T and G to C

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How do we get the DNA out?

EXTRACTION PROCEDURES: Release from cell Isolate and purify

DNA EXTRACTION

Kendall/Hunt Publishing Company 8

Chapter 11

Why such a strange structure?

It's a molecular Xerox!


http://www.youtube.com/watch?v=4jtmOZaIvS0

Chapter 11

We Are the World

We are the World


http://www.youtube.com/watch?v=k2W4-0qUdHY


PCR—PolymeraseChain Reaction

PCR is a technique used for making copies of a defined segment of a DNA molecule. This can be valuable when the amount of evidence is minimal. Millions of copies of DNA can be made from a single speck of blood.


Advantages of PCR Tiny amounts of DNA may be tested Degrade DNA may be tested. Large numbers of copies of specific DNA

sequences at different regions of DNA (loci) can be amplified simultaneously with multiplex PCR reactions.

Commercial kits are now available for easy PCR reaction setup and amplification.

Contaminant DNA, such as fungal and bacterial sources, will not amplify because human-specific primers are used. However, human contamination can be a problem.


DNA Interactive

The website below has an STR animation demonstration. Click on human identification, profiling and then on the third circle called Today’s DNA Profiling to see the demonstration.

Early DNA typing to Today's DNA typingPCR lab simulation


PCR—Polymerase Chain Reaction Procedure

Mix DNA, polymerase, nucleotides Heat to denature...separate strands Cool to anneal the primer...target area of

interest (loci) Heat to extend...copy Repeat 30 times for about one billion

copies! Each cycle takes about 2 minutes and

doubles the number of copies

Chapter 11

Something to sing about...

THE PCR SONG


Chapter 11

The PCR Song by Scientists for Better PCR

There was a time when to amplify DNA,You had to grow tons and tons of tiny cells.(Oooh) Then along came a guy named Dr. Kary Mullis,Said you can amplify in vitro just as well.

Just mix your template with a buffer and some primers,Nucleotides and polymerases too.Denaturing, annealing, and extending,Well it’s amazing what heating and cooling and heating will do.

[Chorus]PCR when you need to detect mutation (detect mutation)PCR when you need to recombine (recombine)PCR when you need to find out who the daddy is (who’s your daddy?)PCR when you need to solve a crime (solve a crime)[x2]



DNA Typing

DNA typing is a method in which DNA is characterized by length or sequence differences. Only one-tenth of a single percent of DNA (about 3 million bases) differs from one person to the next. Scientists use these regions to generate a DNA profile of an individual.


Non-Coding Regions

3 percent of the human DNA sequences code for proteins

97 percent is non-coding and is repetitive; repeating the same sequence over and over

50 percent of the human genome has interspersed repetitive sequences. These show great variety between people


Uses of DNA Profiling

To identify potential suspects To exonerate individuals To identify source of biological evidence for

reconstruction of crime To link or exclude individuals To identify crime and casualty victims To establish paternity


DNA TYPING“Fingerprinting”

RFLP—Restriction Fragment Length Polymorphism

PCR—Polymerase Chain Reaction

STR—Short Tandem Repeats

Chapter 11

Two kinds of differences SEQUENCE

• DNA is a long molecule.• DNA is a lung molecule.

GTCAGTCAGGG

GTCCGTCAGGG LENGTH

• DNA is a long long long long molecule.• DNA is a long long molecule.

CATCATCAT

CATCATCATCATCAT



RFLP—Restriction Fragment Length Polymorphisms

Restriction enzymes are used to cut DNA into smaller fragments that can then be separated and characterized for identification Isolate—separate DNA from the cell

Cut—using restriction enzymes to make shorter base strands

Sort—by size using electrophoresis

Probe selected regions of DNA

Analyze—the specific alleles for identification


Short Tandem Repeats (STR)

STR is a method of DNA typing.

STR’s are locations (loci) on the chromosome that contain short sequences of 2 to 5 bases that repeat themselves in the DNA molecule. The advantages of this method are that it provides greater discrimination, requires less time, a smaller sample size, and the DNA is less susceptible to degradation.


DNA Interactive

The website below has a STR animation demonstration. Click on human identification, profiling and then on the third circle called Today’s DNA Profiling to see the demonstration.

http://www.dnai.org/d/index.html



Each person has two STR types at each locus, one inherited from each parent.

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


Electrophoresis A technique used to separate DNA

fragments.

An electrical current is moved through a gel substance causing molecules to sort by size.

The smaller, lighter molecules will move the furthest on the gel.

The fragments can be visualized for characterization.


Electrophoresis

Pipette the DNA.


Electrophoresis

Load DNA into the gel wells.


Electrophoresis

Run the gel.

Observe and compare bands of DNA.

Profiler Plus Allelic Ladders

D3S1358 FGAVWA

AMEL D8S1179 D21S11 D18S51

D5S818 D13S317D7S820

COfiler Allelic Ladders

D3S1358

AMEL

D7S820

D16S539

TH01TPOX CSF1PO

STR Example


Three Possible Outcomes Match—The DNA profile appears the

same. Lab will determine the frequency.

Exclusion—The genotype comparison shows profile differences that can only be explained by the two samples originating from different sources.

Inconclusive—The data does not support a conclusion as to whether the profiles match.


Determining Probability

Databases have been established that determine how often a particular allele on a loci appears in a given population. By increasing the number of alleles on different loci the probability of having two people with the exact combination becomes astronomical.

Chapter 11

Product Rule at its best!

Testing only 4 loci, we get these results• Locus 1: 5 (1/10) 12 (1/50)• Locus 2: 10 (1/100) 20 (1/40)• Locus 3: 6 (1/4) 8 (1/5)• Locus 4: 4 (1/3) 9 (1/60)

Multiply those together, and what do you get?


Chapter 11

7.2 BILLION

1/7.200000000 1/7.2 billion! What is the population of the world? 7 billion And that is with only 4 loci. Most labs test

at least 13!



Types of DNANuclear found in the nucleus constitutes 23 pair of

chromosomes inherited from both parents

each cell contains only one nuclei

Mitochondrial found in the cytoplasm is inherited only from

mother each cell contains

hundreds to thousands of mitochondria

can be found in skeletal remains, degrade, or low quantity samples


Mitochondrial DNA Analysis of mDNA is more:

rigorous time consuming costly than nucleic testing of DNA

mDNA is constructed in a circular or loop 37 genes are involved in mitochondrial

energy generation Is used when nuclear DNA typing is not

possible


FBI’s CODIS DNA Database

Combined DNA Index System

Used for linking serial crimes and unsolved cases with repeat offenders

Launched October 1994

Links all 50 states

Requires >4 RFLP markers and/or 13 core STR markers

Chapter 11

E:\FORENSICS 1011\Forensic Lessons\FORENSIC SCIENCE CH 11 DNA 0809\codis.pdf


Chapter 11

CODIS STATS UPDATE


Chapter 11

PA Senate Bill 775

PA House Bill 713


Chapter 11

Is it worth it? Ask the families of at least 10 victims.

Consider the Grim Sleeper



The Future

Greater automation of the DNA typing process

Use of SNP’s—single nucleotide polymorphism which measures a one nucleotide change or difference from one individual to another. More sites are needed to differentiate between individuals (30 to 50 SNPs to attain the frequencies of the 13 STR loci), but it can be done with robots and automation.


People in the News

Sir Alec Jeffreys is credited with DNA profiling using RFLP. In September of 1984 after years of work, he saw his first series of blots on an X-ray. The technique was first used in forensics, when in 1985 he was asked by police to confirm the rape confession of 17 year old Richard Buckland, who was denying a rape of another young woman. he DNA from Buckland and the DNA taken from the victims eliminated him as a suspect. Jefferys then used samples from other suspects to later convict Colin Pitchfork whose DNA did match.


More about DNA

For additional information about DNA and some famous cases, check out Court TV’s Crime Library at:

www.crimelibrary.com/criminal_mind/forensics/dna/1.html


Short Tandem Repeats (STR) Procedure

Extract the DNA

Amplify the sample by means of PCR

Separate by electrophoresis

Examine the distance the STR migrates to determine the number of repeats


Mullis Interview

The website below has an interview of Nobel Prize winner, Dr. Kary Mullis, complete with some deep advice for students from one of the century’s greatest minds.

http://nobelprize.org/mediaplayer/index.php?id=428

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