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RFLP technique and interpretation of DNA band patterns

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RFLP technique and interpretation of DNA band patterns. Polymerase Chain Reaction (PCR). RFLP Vs. PCR. Short Tandem Repeat (STR). Nuclear Vs. Mitochondrial DNA. – PowerPoint PPT presentation

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Title: RFLP technique and interpretation of DNA band patterns


1
The History of DNA Forensics
2
What is DNA?
  • DNA is the chemical substance which makes up our
    chromosomes and controls all inheritable traits
    (eye, hair and skin color)
  • DNA is different for every individual except
    identical twins
  • DNA is found in all cells with a nucleus (white
    blood cells, soft tissue cells, bone cells, hair
    root cells and spermatozoa)
  • Half of a individuals DNA/chromosomes come from
    the father the other half from the mother.

3
DNA Review
  • DNA is a double-stranded molecule.
  • The DNA strands are made of four different
    building blocks.
  • An individuals DNA remains the same throughout
    life.
  • In specific regions on a DNA strand each person
    has a unique sequence of DNA or genetic code.

4
Repeated DNA Sequences
  • VNTRs--Tandemly repeated DNA sequences.
  • Interspersed Transposable Elements
  • SINES
  • LINES

5
Cutting Variable DNA Sections
  • Father DNA is a long,long,long long molecule
    that is tightly wound.
  • Mother DNA is a long,long molecule that is
    tightly wound.
  • Restriction Enzymes are proteins that cut DNA
    molecules at specific cut sites.

6
The History of Forensic DNA Analysis Resembles a
War.
  • Laboratories
  • Legal System (Prosecutors / Defense Attorneys)
  • Media Coverage

7
Conventional Blood Typing
  • Used for more than 50 years
  • Utilized ABO blood typing groups
  • Identified genetic variations in blood proteins,
    tissue specific proteins and serum protein types
  • Major problem is that the conventional blood
    protein markers are not found in semen.

8
Development of DNA Analysis Techniques (1970s)
  • RFLP (Restriction Fragment Length Polymorphism)
  • Southern Blot

9
Restriction Fragment Length Polymorphism
  • Restriction Enzymes (biological catalysts) cut
    DNA whenever they encounter a specific DNA
    sequence.
  • Gel electrophoresis separates the fragments of
    DNA according to their length.

10
Size Separation of DNA by Gel Electrophoresis
  • Total DNA Gel
    Electrophoresis
  • Restriction
  • Enzymes

11
Southern Blot
  • A short segment of DNA that is complementary to a
    portion of the desired DNA fragments is labeled
    with a radioactive atom.
  • This probe binds to the fragment of interest on
    the gel electrophoresis.
  • Visualized using X-ray film.

12
A Schematic Representation of RFLP and Southern
Blot of a Single-locus VNTR
13
History of DNA Analysis (1980s)
  • In 1980 David Botstein and others used RFLP to
    construct a human gene map.
  • Used genetic variations as markers

14
Kary Mullis Invented PCR Methods (1984)
  • Polymerase Chain Reaction amplifies short
    specific regions of DNA
  • PCR is an in vitro technique that can yield
    millions of copies of desired DNA
  • Does not use radioactivity

15

16
PCR Amplifies DNA Exponentially
17
In 1984, Alec Jeffreys developed DNA
Fingerprinting.
  • Was searching for disease markers
  • Applied the technique to personal identification
  • Demonstrated that the DNA could be retrieved from
    old dried blood stains
  • Applied the technique to high-profile forensic
    tests

18
A Typical DNA Profile
19
The Pitchfork Case (1986 1987)
  • Jeffreys was consulted in the case of the murder
    and rape of two British schoolgirls.
  • Suspect cleared from analysis of semen samples at
    the scene and blood samples from the suspect.
  • Blooded 4583 men
  • Analysis of blood sample from Colin Pitchfork
    provided a match.

20
Pennsylvania v Pestinikas (1986)
  • First PCR case done in the United States
  • Involved allegations of switching body parts at a
    funeral home

21
Commercial Development of Forensic DNA Testing in
the United States
  • Lifecodes CorporationFounded in 1982 in
    Valhalla, NY as diagnostic company. Began
    performing forensic DNA testing in 1987
  • CellmarkThe US branch of Britains Imperial
    Chemical Industries opened in Germantown, MD in
    1987. Performed the testing for the prosecutor
    in the Simpson case.

22
Trial of Accused Rapist Tommy Lee
Andrews November 3, 1987
  • First case in the U.S. to identify a criminal by
    DNA
  • Trial held in Orlando, Florida
  • A scientist from Lifecodes and a biologist from
    M.I.T. testified that semen left on the victim
    matched Andrews DNA (1 in 10 billion)
  • November 6, 1987 jury returned a guilty verdict.

23
The two major private companies raced each other
to the courtroom.
  • In a shroud of secrecy and an environment of
    expediency they disregarded the usual methods of
    testing new scientific methods
  • Publication and Peer Review
  • Standardization
  • Replication
  • Evaluation of Performance

24
DNA analysis was considered an infallible
prosecution tool.
  • You cant argue with science. a juror in
    Queens
  • DNA evidence is the single greatest advance in
    the search for truth since the advent of
    cross-examination. Judge Joseph Harris of
    Albany, NY

25
DNA analysis was considered an infallible
prosecution tool.
  • In rape cases, when the semen has been matched
    with the defendants and the chance that it came
    from another person is 33 billion to 1, you dont
    need a jury. Robert Brower, defense attorney.

26
New York v Castro
  • First successful defense against DNA analysis
  • Jose Castro, janitor in a nearby building, was
    accused of the stabbing deaths of Vilma Ponce and
    her two-year old daughter, Natasha.

27
New York v Castro
  • Lifecodes Corporation reported that the blood
    found on Castros watch matched that of Vilma
    Ponce with a frequency of 1189,200,000 in the
    Hispanic population.
  • Defense mounted the first successful attempt to
    have DNA evidence excluded.

28
New York v Castro
  • Two defense and two prosecution witnesses agreed
    that Lifecodes had failed to use generally
    accepted scientific techniques. Their data was
    poor and they did not follow procedures for
    interpreting the data
  • Castro later confessed and pled guilty to the
    murders.

29
The FBI and The Royal Canadian Mounted Police Set
Up DNA Laboratories
  • 1987 FBI with NIH began collaborative research to
    establish DNA identification techniques
  • In late 1988 FBI set up their own laboratory at
    their Pennsylvania Avenue headquarters
  • RCMP also set up their own DNA laboratory.

30
FBI Worked With Pioneers in the Field
  • They used four different DNA probes
  • GeneLex
  • Dr. Raymond White of Howard Hughes Medical Center
  • Lifecodes
  • Cellmark

31
  • DNA probes and primers are the key patented
    biomolecules used to identify the individual
    genetic variability.

32
The FBI and RCMP brought standardization to the
indusdry.
  • Established detailed laboratory protocols
  • Performed validation studies
  • Cut through the competitive nature that clouded
    the environment of the testing methods and tools

33
Defense Strategy
  • In 1989, National Association of Criminal Defense
    Lawyers (NACDL) set up a DNA Task Force.
  • Headed by Barry Scheck, a professor at Benjamin
    N. Cardozo Law School and Peter Neufeld, a
    private attorney in Manhattan

34
Defense Strategy
  • Tried to reopen all the convictions involving
    evidence processed by Lifecodes
  • Escalated the conflict surrounding DNA testing
  • Launched a public relations campaign critical of
    DNA typing

35
In 1990 Scheck and Neufeld Prepare for United
States vs Yee.
  • Three members of Hells Angels motorcycle gang of
    Cleveland Ohio were accused of killing David
    Hartlaub thinking he was a member of a rival gang
  • The victim was shot 14 times with a MAC 10
    machine gun.

36
Blood Evidence on the Scene
  • Most was determined to be that of the victim
  • Some belonged to one of the defendants
  • Theorya ricocheting bullet had hit one of the
    suspects.

37
Prosecution Expert Witnesses
  • Thomas Caskey, Baylor College of Medicine
  • Kenneth Kidd, Yale geneticist
  • Bruce Budowle, FBI DNA scientist

38
Defense Expert Witnesses
  • Richard Lewontin, geneticist from Harvard
  • Daniel Hartl, geneticist at Washington University
  • Eric Lander, mathematician and geneticist at
    M.I.T.

39
Defense Argued
  • FBIs published articles on matching criteria
    were ambiguous and inconsistent.

40
Prosecution Countered
  • In spite of disputes over match criteria, the
    multi-probe match produced a highly likelihood
    that the specimens came from two different
    people.

41
United States vs Yee
  • DNA matches were admitted as evidence at the
    trial and at the appeal.
  • The documents from the FBI would later find their
    way into the courtroom.

42
The National Academy of Sciences National
Research Council (NCR) Report
  • A federal study intended to be the definitive
    study on the problems of forensic DNA
  • The most contended issue--How to calculate
    statistical probability
  • The frequencies of sub-groups could differ widely
    from that in the larger population

43
NCR ReportDNA Technology in Forensic Science
(1992) Criticized
  • Ceiling principle
  • No population geneticists or staticians on the
    panel
  • Inconsistencies
  • Did not address paternity cases or PCR

44
NCR ReportDNA Technology in Forensic Science
  • The report was to be the final word in clarifying
    the role of DNA forensics.
  • This document is often cited by defense lawyers
    in arguing of the exclusion of DNA evidence.

45
The Media and DNA Forensics
  • First the Media proclaimed that the new
    technology was a miracle.
  • It recorded its glowing victories.
  • Followed by giving opponents to DNA analysis a
    venue for airing objections and charges (many of
    which were unfounded and unchallenged)

46
The Media and DNA Forensics
  • The Washington Post headline read, Panel Backs
    DNA Tests as Crime Evidence.

47
Nature Published Articles Exploring Forensic DNA
  • Alec Jeffreys announced his methods in Nature.
  • DNA fingerprinting dispute laid to rest Budowle
    and Lander Oct. 27, 1994

48
  • Lander and Budowle declare that after 400
    technical papers, 100 scientific conferences,
    three sets of DNA analysis guidelines, 150 court
    cases, and an exhaustive three-year study by the
    National Research CouncilThe DNA fingerprinting
    wars are over.

49
1994
  • National Research Council reconvened to try to
    rectify the deficiencies of their first report.
  • The trial of the century convened Dollars v
    DNA or California v OJ Simpson.

50
Summary
  • Forensic DNA testing was developed rapidly by
    short-sighted commercial interests
  • Standards were not developed as quickly as
    necessary
  • Prosecution oversold DNA evidence
  • Expert witnesses had a vested interest in fueling
    the controversy.

51
Summary
  • Sensational and inaccurate media coverage spread
    misinformation about DNA.
  • The judicial system is sometimes indifferent to
    choices that could expedite justice.

52
The Media and DNA Forensics
  • The DNA Technology in Forensic Science report
    strongly endorsed the continued use of DNA typing
    in the courts.
  • The New York Times front page article headline
    read U.S. Panel Seeking Restriction on Use of
    DNA in Courts.

53
Where is Forensic DNA Analysis Today?
  • Forensic labs have implemented a process of peer
    review, self-regulation and accreditation
  • DNA analysis has freed over 156 convicts. The
    Innocence Project at Yeshiva University is run by
    Barry Sheck and Peter Neufeld.

54
Where is Forensic DNA Analysis Today?
  • Paternity testing.
  • Historical case of Thomas Jefferson as the likely
    father of children of slave, Sally Hemmings.
  • Resolved questions surrounding the disputed
    descendent of the Czar Nicholas II

55
Terms to Know
  • ABO Blood Typing
  • RFLP
  • Southern Blot
  • PCR
  • Kary Mullis
  • Alec Jeffreys
  • DNA Fingerprinting
  • DNA typing
  • DNA profiling
  • Probe
  • Primer

56
Resources
  • DNA in the Courtroom Coleman Swenson, GeneLex
    Press 1994.
  • DNA Technology in Forensic Science Committee on
    DNA Technology in Forensic ScienceNational
    Research Council National Academy Press 1992

57
Ch 13 - DNA
  • Nucleotides and DNA.
  • Double helix.
  • Base pairing and double helix.
  • Sequence of bases in DNA and synthesis of
    proteins.
  • DNA replication and its significance to forensic
    science.
  • Recombinant DNA technology.

58
  • DNA strands that code for protein synthesis Vs.
    strands that contain repeated sequence of bases.
  • Restriction Fragment Length Polymorphism (RFLP).
  • RFLP technique and interpretation of DNA band
    patterns.
  • Polymerase Chain Reaction (PCR).
  • RFLP Vs. PCR.
  • Short Tandem Repeat (STR).
  • Nuclear Vs. Mitochondrial DNA.

59
  • DNA computerized data base in criminal
    investigation.
  • Preservation of blood stains for DNA analysis.
  • http//www.biology.washington.edu/fingerprint/dnai
    ntro.html
  • http//www.biology.arizona.edu/human_bio/problem_s
    ets/DNA_forensics_1/DNA_forensics.html
  • http//whyfiles.org/014forensic/genetic_foren.html
  • http//whyfiles.org/014forensic/genetic_foren2.htm
    l
  • http//www.fbi.gov/hq/lab/handbook/examsdna.htm
  • http//arbl.cvmbs.colostate.edu/hbooks/genetics/me
    dgen/dnatesting/dnatest_tech.html

60
  • DNA abbreviation for deoxyribonucleic acid- the
    molecules carrying the bodys genetic
    information. DNA is double-stranded in the shape
    of a double helix
  • Chromosome a rodlike structure in the cell
    nucleus, along witch the genes are located. It
    is composed of DNA surrounded by other material,
    mainly proteins
  • Polymer a substance composed of a large number
    of atoms. These atoms are usually arranged in
    repeating units, or monomers

61
  • Nucleotide the unit of DNA consisting of one of
    four bases-adenine, guanine, cystine, or
    thymine-attached to a phosphate sugar group
  • Proteins polymers of amino acids that play
    basic roles in the structures and functions of
    living things
  • Amino acids the building blocks of proteins.
    There are 20 common amino acids. Amino acids are
    linked together to form a protein. They types of
    amino acids and the order in which theyre linked
    determine the character of each protein

62
  • Human Genome the total DNA context found within
    the nucleus of a cell. In humans, it is
    composed of approximately 3 billion base pairs of
    genetic information
  • Replication the synthesis of new DNA form
    existing DNA
  • Polymerase Chain Reaction (PCR) a technique for
    replicating or copying a portion of a DNA strand
    outside a living cell. This technique leads to
    millions of copies of the DNA strand

63
  • Restriction Enzymes chemicals that act as
    scissors to cut DNA molecules at specific
    locations
  • Restriction Fragment Length Polymorphisms (RFLP)
    different fragment lengths of base pairs that
    result from cutting a DNA molecules with
    restriction enzymes
  • Hybridization the process of joining two
    complementary strands of DNA together to form a
    double-stranded molecule

64
  • Short Tandem Repeats (STRs) regions of a DNA
    molecule that contain short segments consisting
    of three to seven repeating base pairs
  • Multiplexing a technique that simultaneously
    detects more than one DNA marker in a single
    analysis
  • Amelogenin gene a genetic locus found useful
    for determining gender

65
  • Mitochondria small structures located outside
    the nucleus of a cell. These structures are
    responsible for supplying energy to the cell.
    Maternally inherited DNA is found in each
    mitochondria
  • Sequencing a procedure used to determine the
    order of the base pairs that comprise DNA
  • Substrate control an unstained object adjacent
    to an area upon which biological material had
    been deposited
  • Buccal cells cells derived from the inner cheek
    lining

66
Ch. 13 - Forensic DNA
67
The Cell
  • The smallest unit of life
  • The nucleus is the brain of the cell
  • contains all the genetic info the cell needs to
    exist to reproduce
  • In most types of cells, genetic information is
    organized into structures called chromosomes

68
Chromosomes
  • In most types of cells, genetic information is
    organized into structures called chromosomes
  • usually X shaped
  • Y chromosome in males
  • 23 pairs in humans
  • one from mother one from father

69
Genes
  • Each chromosome contains hundreds to thousands
    information blocks called genes
  • Each gene is the blueprint for
    a specific type of protein in the body
  • only identical twins will have all the genes
    identical

70
Chromosomes
  • Each chromosome is a single polymeric molecule
    called DNA
  • if fully extended the molecule would be about 1.7
    meters long
  • unwrapping all the DNA in all your cells
  • cover the distance from earth to moon 6,000 times

71
Structure of DNA
72
Nucleotides
  • DNA is a polymer built from monomers called
    nucleotides
  • Each nucleotide is consists of
  • deoxyribose
  • pentose sugar
  • phosphoric acid
  • a nitrogenous base

73
The phosphate
The sugar
74
Nitrogenous Bases
  • The Purines
  • Adenine (A)
  • Guanine (G)

75
Nitrogenous Bases
  • The Pyrimidines
  • Cytosine (C)
  • Thymine (T)

76
The DNA Backbone
  • The monomers are linked together by
    phosphodiester bridges (bonds)
  • links the 3 carbon in the ribose of one
    nucleotide to the 5 carbon in the ribose of the
    adjacent nucleotide

77
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78
The DNA Double Helix
  • DNA is normally a double stranded macromolecule
  • Two polynucleotide chains are held together by
    H-bonding
  • A always pairs with T
  • C always pairs with G

79
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80
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81
  • 5 T-T-G-A-C-T-A-T-C-C-A-G-A-T-C 3
  • 3 A-A-C-T-G-A-T-A-G-G-T-C-T-A-G 5
  • In a double helix the strands go in opposite
    directions

82
Functions of DNA
  • Two Functions
  • To transmit information from one generation of
    cells to the next
  • To provide the information for the synthesis of
    components (proteins) necessary for cellular
    function

83
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84
DNA Fingerprinting
  • The basic structure of everyones DNA is the same
  • the difference between people is the ordering of
    the base pairs
  • Every person can be distinguished by the sequence
    of their base pairs
  • millions of base pairs make this impractical
  • a shorter method uses repeating patterns that are
    present in DNA

85
VNTRs
  • DNA strands contain information which directs an
    organisms development
  • exons
  • Also contain stretches which appear to provide no
    relevant genetic information
  • introns
  • repeated sequences of base pairs
  • Variable Number Tandem Repeats (VNTRs)
  • can contain anywhere from 20 to 200 base pairs

86
VNTRs
  • All humans have some VNTRs
  • VNTRs come from the genetic information donated
    by parents
  • can have VNTRs from mother, father or a
    combination
  • will not have a VNTR that is from neither parent

87
D1 biological daughter of both parents D2
child of mother former husband S1 couples
biological son S2 adopted son
88
VNTR Analysis
  • Usually an individual will inherit a different
    variant of the repeated sequence from each parent

89
VNTR Analysis
  • PCR primers bracket the locus
  • PCR reaction forms a nucleotide chain from the
    template

90
VNTR Analysis
  • The length of the amplified DNA its position
    after electrophoresis will depend on the number
    or repeated bases in the sequence

91
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92
Analysis used 3 different VNTR loci for each
suspect giving 6 bands
93
Although some individuals have several bands in
common, the overall pattern is distinctive for
each
94
Suspects A C can be eliminated B remains a
suspect
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