1. Chapter 11 DNA Analysis “The capacity to blunder slightly is the
real marvel of DNA.
Without this special
attribute, we would still
be anaerobic bacteria and
there would be no music.”
—Lewis Thomas, Physician, author

2. Kendall/Hunt Publishing Company
DNA Analysis
Students will learn:
That DNA is a long-chain polymer found in nucleated cells, which contain 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 RFLP, PCR, and STRs to characterize DNA.
The role that statistics plays in determining the probability that two people would have the same sequence in a fragment of DNA.
Kendall/Hunt Publishing Company

3. Kendall/Hunt Publishing Company
DNA Analysis
Students will be able to:
Explain that DNA is a long molecule, tightly packed in the form of a chromosome with genetic material wrapped around it.
Isolate and extract DNA from cells.
Describe the function and purpose of a restriction enzyme.
Calculate probabilities of identity using STR.
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
1998—FBI launches CODIS database
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 gentlemen 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:
Kendall/Hunt Publishing Company

6. 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
Kendall/Hunt Publishing Company

7. Kendall/Hunt Publishing Company
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
Most abundant in buccal (cheek) cells
Red blood cells have no nuclei; and therefore, no nuclear DNA
DNA obtained from blood comes from white blood cells
Kendall/Hunt Publishing Company

8. Kendall/Hunt Publishing Company
Blood Cells
Saliva
Skin Cells
Hair Root
Sperm
Fingernail
Kendall/Hunt Publishing Company

9. Kendall/Hunt Publishing Company
Blood
At one time, blood at a crime scene was significant to investigators for its presence alone — bloodstains and spatters often tell their own story of events. But today scientists can perform further tests, including blood typing and DNA analysis, that can help identify the victim or offender. Pictured here is a color-enhanced image of blood cells and platelets, magnified over 5,200 times by a scanning electron microscope.
Kendall/Hunt Publishing Company

10. Kendall/Hunt Publishing Company
Saliva
Investigators can extract DNA from saliva left behind on items such as cigarettes, envelopes or drinking straws at the crime scene to help identify their killer or victim. One suspect was connected to the World Trade Center bombing, for example, after authorities matched his DNA with that from saliva on an envelope delivered by one of the conspirators. This saliva has been magnified 40 times.
Kendall/Hunt Publishing Company

11. Kendall/Hunt Publishing Company
Sperm
In cases of rape and other sexual crimes, the seminal fluid left by the attacker can provide important evidence. Since sperm remains alive for only a short period of time, its condition can indicate the time of the attack; such samples can also provide information on blood type and now identity, through DNA. Pictured here is a color-enhanced image of sperm, magnified over 3,000 times by a scanning electron microscope.
Kendall/Hunt Publishing Company

12. Kendall/Hunt Publishing Company
Fingernail
While their utility is still under debate, fingernails, like fingerprints, are thought to be unique and thus useful in linking suspects to crime scenes. This is because the underside of fingernails and toenails have grooves, called striations, that form a pattern unique to each finger or toe; nails from one individual match up to their owner much like bullets fired from the same gun. Fingernail evidence has been used in several U.S. cases since the mid-1970s. Pictured here is a section of fingernail magnified over 300 times by a scanning electron microscope.
Kendall/Hunt Publishing Company

13. Kendall/Hunt Publishing Company
Hair Root
Forensic scientists can analyze the DNA found in the cells of a single hair root to identify their killer or victim. Investigators often find hairs with roots attached at crime scenes, indicating a struggle took place. Pictured here is a color-enhanced image of a hair root, magnified over 90 times by a scanning electron microscope.
Kendall/Hunt Publishing Company

14. Kendall/Hunt Publishing Company
Human Skin
DNA can sometimes be obtained from skin cells that an attacker unwittingly leaves beneath the fingernails of a victim during a fight. In one case, police found a victim with hair clutched in her hand and skin under her nails; investigators were able to use this evidence to link her husband to the crime. Pictured here is a color-enhanced image of a healthy skin surface, magnified nearly 1,000 times by a scanning electron microscope.
Kendall/Hunt Publishing Company

15. Kendall/Hunt Publishing Company
DNA Typing
DNA typing is a method in which DNA is converted into a series of bands that ultimately distinguish each individual. 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.
Kendall/Hunt Publishing Company

16. Kendall/Hunt Publishing Company
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
Kendall/Hunt Publishing Company

17. Kendall/Hunt Publishing Company
Uses of DNA Profiling
To identify potential suspects
To exonerate individuals
To identify crime and casualty victims
To establish paternity
To match organ donors
Kendall/Hunt Publishing Company

18. DNA TYPING “Fingerprinting”
RFLP—Restriction Fragment Length Polymorphism
PCR—Polymerase Chain Reaction
STR—Short Tandem Repeats
Kendall/Hunt Publishing Company

19. 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
Analyze—the specific alleles for identification
Kendall/Hunt Publishing Company

20. PCR—Polymerase Chain 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.
Kendall/Hunt Publishing Company

21. PCR—Polymerase Chain Reaction Procedure
Heat the DNA strands, causing the strands to separate (unzip).
Cool the mixture and add a primer, a short sequence of base pairs that will add to its complementary sequence on the DNA strand.
Finally, add a DNA polymerase and a mixture of free nucleotides to the separated strands. Heat again to around 75° C for the completion.
Kendall/Hunt Publishing Company

22. PCR—Polymerase Chain Reaction
The outcome is a doubling of the number of DNA strands. Heating, cooling, and strand rebuilding is repeated typically 25 to 30 times, yielding more than one million copies of the original DNA molecule. Each cycle takes less than two minutes from start to finish.
Kendall/Hunt Publishing Company

23. Kendall/Hunt Publishing Company
Advantages of PCR
Minute amounts of DNA may be used for amplification.
DNA degraded to fragments only a few hundred base pairs in length can serve as effective templates for amplification.
Large numbers of copies of specific DNA sequences 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.
Kendall/Hunt Publishing Company

24. Kendall/Hunt Publishing Company
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.
After developing, the fragments can be visualized for characterization.
animation
Kendall/Hunt Publishing Company

25. Kendall/Hunt Publishing Company
Electrophoresis
Pipette the DNA.
Kendall/Hunt Publishing Company

26. Kendall/Hunt Publishing Company
Electrophoresis
Load DNA into the gel wells.
Electrophoresis video
Kendall/Hunt Publishing Company

27. Kendall/Hunt Publishing Company
Electrophoresis
Run the gel.
Observe and compare bands of DNA.
Kendall/Hunt Publishing Company

28. Kendall/Hunt Publishing Company
X-ray of results
Kendall/Hunt Publishing Company

29. Short Tandem Repeats (STR)
STR is another 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.
Kendall/Hunt Publishing Company

30. Kendall/Hunt Publishing Company
Karyotype- show number and appearance of an organism’s chromosomes. Genes are located on the chromosomes.
Kendall/Hunt Publishing Company

31. Kendall/Hunt Publishing Company

32. Short Tandem Repeats (STR) Procedure
Extract the gene TH01 from the sample. (TH01 has seven human variants with a repeating sequence of A-A-T-G)
Amplify the sample by means of PCR
Separate by electrophoresis
Examine the distance the STR migrates to determine the number of times TH01 repeats
Kendall/Hunt Publishing Company

33. Short Tandem Repeats (STR)
Each person has two STR types for TH01—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 person.
Kendall/Hunt Publishing Company

34. 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.
Kendall/Hunt Publishing Company

35. Profiler Plus Allelic Ladders
D3S1358
VWA
FGA
AMEL
D8S1179
D21S11
D18S51
D5S818
D13S317
D7S820

36. COfiler Allelic Ladders
D3S1358
D16S539
AMEL
TH01
TPOX
CSF1PO
D7S820

37. STR Example

39. 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 miniscule.
Kendall/Hunt Publishing Company

40. Kendall/Hunt Publishing Company
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.
Kendall/Hunt Publishing Company

41. Kendall/Hunt Publishing Company
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.
Kendall/Hunt Publishing Company

42. Kendall/Hunt Publishing Company
Types of DNA
Nuclear
found in the nucleus
constitutes 23 pairs 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
Nuclear DNA is present in the head of the sperm. Mitochondrial DNA is present in the tail. At conception, the head of the sperm enters the egg and unites with the nucleus. The tail falls off, losing the father’s mitochondrial DNA.
Kendall/Hunt Publishing Company

43. Kendall/Hunt Publishing Company
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
Kendall/Hunt Publishing Company

44. Kendall/Hunt Publishing Company
FBI’s CODIS DNA Database
Combined DNA Index System
Used for linking serial crimes and unsolved cases with repeat offenders
Launched October 1998
Links all 50 states
Requires >4 RFLP markers and/or 13 core STR markers
Kendall/Hunt Publishing Company

45. Kendall/Hunt Publishing Company
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.
Kendall/Hunt Publishing Company

46. Kendall/Hunt Publishing Company
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. The 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.
Kendall/Hunt Publishing Company

47. Kendall/Hunt Publishing Company
More about DNA
For additional information about DNA and some famous cases, check out Court TV’s Crime Library at:
Kendall/Hunt Publishing Company