Download DNA Analysis Chapter 11

DNA Analysis
Chapter 11
©2010 Elsevier, Inc.
INTRODUCTION
• Serology has several drawbacks
– Lack of stability of many proteins and limited
ability to discriminate among a population of
people
• DNA has increased understanding of the
development of plants, animals and diseases
– Profound impact on forensic science
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THE NATURE OF DNA
• DNA (deoxyribonucleic acid) is a molecule
found in nearly all cells
– DNA is a polymer, a molecule made up of
repeating simpler units, called monomers
– Located in nucleus and mitochondria
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Nuclear DNA
– Structure is a double helix
• A helix is a spiral-shaped object
• Sugar molecules and phosphates connect the helices
– Four base nucleotides: adenine (A), guanine (G), cytosine (C),
and thymine (T) dangle off of each sugar molecule
– A and T will bond with one another
– C and G will bond with one another
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Nuclear DNA
– DNA molecule consists of sugar-phosphate
backbones connected by linked base pairs: A-T, TA, G-C, C-G
• Order of these pairs is controlled by genetic code, or
blueprint that determines the characteristics of a
person
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DNA in Cells
– Most cells have a nucleus, where most of cell
functions are controlled
– Within nucleus, DNA is arranged into 46 structures
called chromosomes
• Chromosomes are arranged into 23 pairs
– One of each pair comes from father’s sperm and the other
from the mother’s egg
– One pair of chromosomes determines the sex of the individual
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DNA in Cells
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Genes and the Genetic Code
• Genes are sections within the long strands of
DNA
– Ordering of base pairs in genes provides chemical
instructions to manufacture particular proteins in the
body
– Genetic instructions are copied onto RNA (ribonucleic
acid), which transmits this information to protein
manufacturing sites within the cells
• More than 99% of human DNA is exactly the
same, less than 1% differentiates one human
being from another
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Variations of Genes: Alleles
– Some traits are determined by a single gene on one
chromosome; others are determined by multiple
genes at several locations
• If a person inherits the same form of a gene from the
mother and the father, the person is said to be homozygous
• If a person inherits different forms of the same gene, the
person is said to be heterozygous
• Different forms of the same gene or other DNA fragment at
the same locus are called alleles
• Some alleles are dominant and some are recessive
– Variation is basis for DNA profile
• The visible manifestation of variability of alleles is called
phenotype
• Genotype is the genetic description of the allele
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Variations of Genes: Alleles
– In DNA analysis, loci that are polymorphic are
purposely chosen
• More discriminating analysis
– Two types of variability in alleles
• Sequence polymorphism: Two sequences of double
stranded DNA are exactly the same except at one
location
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Variations of Genes: Alleles
• Length polymorphism: Consists of a series of base pairs
that are repeated
– Base pairs that repeat without any intervening pairs are
referred to as tandem repeats
– When variation in the number of repeats occurs from one
individual to the next, the locus is described as having a
variable number of tandem repeats (VNTR)
– DNA type is a description of the types of alleles at all of the
locations being analyzed on the genome
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Population Genetics
• Science of population genetics answers
questions about DNA typing and likelihood of
matching DNA
– Frequency of occurrence for alleles analyzed are
determined
– Multiply the frequency of occurrence for each
allele to determine the frequency of occurrence of
all of these alleles at one time
• Technique of multiplying probabilities together is called
the product rule
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Population Genetics
• To determine an overall DNA type, each data
point must be independent of the other data
points
– Loci used in genotyping have been tested to check
for independence
– Product rule has yielded genotypes that are so
rare that the chances of reoccurrence within the
population is extremely small
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DNA TYPING
• Restriction Fragment Length Polymorphism
(RFLP) was the first DNA typing method to be
widely used by forensic biologists
• Polymerase chain reaction (PCR) is now used
to increase the amount of DNA by
amplification
• Most laboratories use a typing method known
as short tandem repeats (STR)
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Restriction Fragment Length
Polymorphism (RFLP)
– DNA is extracted from biologic material and then
severed into small fragments call minisatellites or
variable number tandem repeats (VNTR) using
restriction enzymes
• Length polymorphism is used to discriminate a
population of people.
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How RFLP Works
– Pieces of DNA are cut out of strand by restriction
enzymes, also known as endonucleases
• Designed to cut DNA at specific sequence of bases
– In RFLP, polymorphic regions of DNA are identified
• Regions are hypervariable; they have a large number of
alleles
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How RFLP Works
– RFLP typing in the US predominantly used single
locus VNTR analysis
• A result of problems with limited or degraded DNA
– Separation of DNA Fragments
• Gel electrophoresis is used to separate fragments of
DNA
– Calibration standards, or ladders, are used to determined the
lengths of the VNTRs when the gel is developed
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How RFLP Works
• A technique known as Southern Blotting is used to
transfer the separated DNA fragments onto a more
rugged nylon membrane
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Visualization of VNTRs
• Probe hybridization is used
to visualize the VNTRs
• Chemiluminescence or
radio-labeled DNA are used
for visualization
– Exposed film is called an
autorad
• VNTR fragments cannot be
viewed as discrete alleles,
rather a band
– Match probabilities can be
calculated
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The Polymerase Chain Reaction (PCR)
• Polymerase chain reaction is used for making
copies of DNA using polymerase enzymes
– Overcame issue of relatively large quantities
required for RFLP
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The PCR Process
– Sensitive to contamination; performed in location
physically isolated from where subsequent
amplifications will be performed
– Three steps to process take place in thermal cycler, an
apparatus capable of achieving and maintaining
preset temperatures very precisely
• Denaturation: The double stranded DNA denatures; bonds
break between the base pairs that hold the strands together
– Result is single stranded DNA
– Strands are templates for formation of a new piece of double
stranded DNA
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The PCR Process
• Annealing: Attach a short strand of
synthetic DNA to each of the separated
strands
– Called primers because they will mark the
starting points for addition of new bases to
complete the reproduction of each strand
• Extension: Single bases, or nucleotides,
are added to the primer
– Strand is built up and a new piece of double
stranded DNA is produced
– Strand is built up and a new piece of
double stranded DNA is produced
– Cycle of three steps is repeated until
sufficient DNA is produced; 25-40 cycles
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The PCR Process
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DNA Typing of PCR Product
– Very sensitive methods are not required, can run a
yield gel experiment on agarose and stain with
ethidium bromide
– Human Leukocyte Antigen (HLA) DQ alpha gene was
the first DNA region widely subjected to amplification
and typing
• DQ alpha and a number of other genes called polymarker
are typed using a method called reverse dot blot
– Involves identifying the particular alleles present by reacting
them with color forming reagents on specially treated nylon strips
– Not often used in forensic science
» Not able to associate sample with just one individual
» Not capable of resolving multiple DNA types in mixtures
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Short Tandem Repeats (STRs)
• STRs are length polymorphic sequences of short
strands of DNA that range from two to six base pairs
long and that repeat in tandem
– Short tandem repeats are called microsatellites
– Longer repeats, typed by RFLP, are called minisatellites
• STR markers exhibit high variability in a population, are
less sensitive to degradation of the DNA, many
microsatellites to choose from
• Combined DNA Index System (CODIS) is a standard
database, containing 13 loci each with four base pair
repeats
– Gender is determined by analyzing the Amelogenin locus
• DNA is amplified by PCR and separated
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STRs
• Capillary electrophoresis is used and DNA is
detected by means of laser-induced
fluorescence
– Results in electropherogram
– Large amount of data for analysis
– Allelic ladders are strands of DNA made up of all
common alleles present at each STR locus and are
used for calibration
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STRs
• FBI decided that any DNA type whose odds of
a chance occurrence exceeded 1,000 times
the U.S. population would be considered
individualized
– All associations expressed as probabilities
– Identical twins have the same DNA, at least
insofar as forensic DNA methods are concerned
– Care should be taken when evaluating such
statistics of rare occurrence
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Gender Identification
– Two approaches to gender identification using
DNA typing
• Amelogenin is a locus on one of the chromosomes and
can be used to determine sex
• Second approach uses Y-STRs to determine gender
– Useful when mixed samples are present
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Mitochondrial DNA (mtDNA)
• Mitochondria are small
structures located within
practically all animal and plant
cells
– Serve as energy mediators of
the cell
• Differences between mtDNA
and genomic DNA
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Mitochondrial DNA (mtDNA)
• Number of differences between mtDNA and genomic DNA
– mtDNA is circular in shape
– Much less mtDNA is present; more copies of mtDNA in
mitochondria cells as compared to few copies of genomic DNA
– mtDNA contains non-coding region, which can be useful for
comparing known and questioned DNA samples
– All mtDNA comes from the mother
– mtDNA shows a high degree of variation between unrelated
people
– mtDNA is useful in typing samples that have low quantities of
DNA, are degraded or are very old
• Most forensic laboratories that do genomic DNA typing also
do mtDNA typing
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INTERPRETATION OF
DNA TYPING RESULTS
• Purity Issues
– Difficulty in interpretation comes from DNA that
has been compromised in some way
• Contamination: Introduction of foreign material
• DNA from more than one source
• Degradation: Break down due to extreme heat, light,
time, humidity, biological causes and presence of
certain chemicals
• Extraneous materials: I.e. clothing dyes, blood
components, soil
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Comparison of DNA Samples
• Rigorous scientific testing has been done that validates
the concepts and testing methodologies for human
DNA typing
• Valid scientifically determined statistical frequencies
for the alleles at the loci that are used in DNA typing
have been calculated
– Statistical methods are used
– Term commonly used to express this comparison is
“match”
• No significant or unexplainable differences between the known
and unknown materials
– Rather than “match” the term “genetic concordance” is
used to describe the relationship between two DNA
samples
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Estimation of Population Frequencies
– Large numbers of people are tested to determine
population frequency
• Number of times each allele appears in population
– Determining population frequency for an entire
DNA profile is not so simple
• Population frequencies of genotypes made up of
multiple loci can be calculated by taking the product of
the population frequencies of each individual locus
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Interpretation of DNA Typing Results:
Purity Issues
• Contamination
– Difficulty in interpretation comes from DNA that has
been compromised in some way
• Contamination: Introduction of foreign material
• DNA from more than one source
• Degradation
– Degradation: Break down due to extreme heat, light,
time, humidity, biological causes and presence of
certain chemicals
– STR analysis is better adapted to degraded DNA than
RFLP
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DNA DATABASE:
THE FBI CODIS SYSTEM
• Combined DNA Index System (CODIS) is the
national database of DNA profiles
administered by the FBI
– Contains thousands of DNA profiles
– Arranged in tiers: local, state, national
– Contains three databases: forensic, criminal
offenders, missing persons
– Data consists of genotypes from 13 STR loci
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DNA Case Backlog
– Many states require some or all people arrested
for crimes to be DNA typed
– Caused huge backlog in forensic science
laboratories
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CHAPTER SUMMARY
• DNA molecule is a double helix containing base pairs
linked to a chemical backbone
• DNA typing methods utilize differences in human
genome to indentify people from biological evidence
– RFLP
– PCR
• CODIS contains nearly six million profiles
• Innocence Project uses DNA typing to re-examine
criminal convictions
• mtDNA is useful for typing old, degraded samples
©2010 Elsevier, Inc.