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“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
Chapter 11 Kendall/Hunt Publishing Company 3
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.
Chapter 11 Kendall/Hunt Publishing Company 4
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
Chapter 11 Kendall/Hunt Publishing Company 5
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:
Chapter 11 Kendall/Hunt Publishing Company 6
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
Chapter 11 Kendall/Hunt Publishing Company 7
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
Chapter 11
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!
Kendall/Hunt Publishing Company 9
Chapter 11
We Are the World
We are the World
Kendall/Hunt Publishing Company 10
Chapter 11 Kendall/Hunt Publishing Company 11
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.
Chapter 11 Kendall/Hunt Publishing Company 12
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.
Chapter 11 Kendall/Hunt Publishing Company 13
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
Chapter 11 Kendall/Hunt Publishing Company 14
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
Kendall/Hunt Publishing Company 15
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]
Kendall/Hunt Publishing Company 16
Chapter 11 Kendall/Hunt Publishing Company 17
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.
Chapter 11 Kendall/Hunt Publishing Company 18
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
Chapter 11 Kendall/Hunt Publishing Company 19
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
Chapter 11 Kendall/Hunt Publishing Company 20
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
Kendall/Hunt Publishing Company 21
Chapter 11 Kendall/Hunt Publishing Company 22
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
Chapter 11 Kendall/Hunt Publishing Company 23
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.
Chapter 11 Kendall/Hunt Publishing Company 24
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
Chapter 11 Kendall/Hunt Publishing Company 25
Chapter 11 Kendall/Hunt Publishing Company 26
Short Tandem Repeats (STR)
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.
Chapter 11 Kendall/Hunt Publishing Company 27
Short Tandem Repeats (STR)
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.
Chapter 11 Kendall/Hunt Publishing Company 28
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.
Chapter 11 Kendall/Hunt Publishing Company 29
Electrophoresis
Pipette the DNA.
Chapter 11 Kendall/Hunt Publishing Company 30
Electrophoresis
Load DNA into the gel wells.
Chapter 11 Kendall/Hunt Publishing Company 31
Electrophoresis
Run the gel.
Observe and compare bands of DNA.
Chapter 11 Kendall/Hunt Publishing Company 32
Profiler Plus Allelic Ladders
D3S1358 FGAVWA
AMEL D8S1179 D21S11 D18S51
D5S818 D13S317D7S820
COfiler Allelic Ladders
D3S1358
AMEL
D7S820
D16S539
TH01TPOX CSF1PO
STR Example
Chapter 11 Kendall/Hunt Publishing Company 37
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.
Chapter 11 Kendall/Hunt Publishing Company 38
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?
Kendall/Hunt Publishing Company 39
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!
Kendall/Hunt Publishing Company 40
Chapter 11 Kendall/Hunt Publishing Company 41
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
Chapter 11 Kendall/Hunt Publishing Company 42
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
Chapter 11 Kendall/Hunt Publishing Company 43
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
Kendall/Hunt Publishing Company 44
Chapter 11
CODIS STATS UPDATE
Kendall/Hunt Publishing Company 45
Chapter 11
PA Senate Bill 775
PA House Bill 713
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Chapter 11
Is it worth it? Ask the families of at least 10 victims.
Consider the Grim Sleeper
Kendall/Hunt Publishing Company 47
Chapter 11 Kendall/Hunt Publishing Company 48
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.
Chapter 11 Kendall/Hunt Publishing Company 49
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.
Chapter 11 Kendall/Hunt Publishing Company 50
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
Chapter 11 Kendall/Hunt Publishing Company 51
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
Chapter 11 Kendall/Hunt Publishing Company 52
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