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American Society of Cytopathology Core Curriculum in Molecular Biology Copyright 2010 American Society of Cytopathology
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American Society of Cytopathology Core Curriculum in Molecular Biology
Chapter 5
Applications of Molecular Testing
Molecular Tests for Identity
Stephanie A. Hamilton, EdD, SCT, MB(ASCP)
MD Anderson Cancer Center
Houston, Texas
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Timeline of Identity Testing
1800s
1900
1960
1980s
1985
1990s
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Fingerprint
ABO blood group typing
HLA typing
DNA analysis (VNTRs)
First DNA “fingerprint” used in court case
DNA analysis (STRs)
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Mutations and Polymorphisms
• Transmissible (inheritable) change in DNA sequence is a mutation or polymorphism which may or may not produce phenotypic differences
• Mutation: a DNA sequence change present in a relatively small proportion of a population
• Polymorphism: a DNA sequence change present in at least 1% of a population
Variation among individuals of noncoding (does not code for a protein), repetitive sequences in DNA is key to identity testing
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Types of Polymorphisms
• Single Nucleotide Polymorphisms (SNPs)
– A single nucleotide difference in a genomic sequence
– Example: Human leukocyte antigen (HLA) locus. This is a highly polymorphic region where SNPs occur frequently. Variability codes for peptides that establish self‐identity of the immune system. Used for determining compatibility of transplant donors.
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Types of Polymorphisms
• Long Interspersed Nucleotide Sequences (LINES)
– Are highly repeated sequences
– 6‐8 kbp in length
– Contain RNA polymerase promoters
– Contain open reading frames related to reverse transcriptase of retroviruses
– More than 500,000 LINES in genome
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Types of Polymorphisms
• Short Interspersed Nucleotide Sequences (SINES)
– 0.3 kbp in length
– More than 1,000,000 copies per genome
– Include Alu elements (named for having recognition sites for the ALuI restriction enzyme)
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Types of Polymorphisms
• Variable Number Tandem Repeat (VNTR)
– A location in genome where a short nucleotide sequence is organized as a tandem repeat (clustered together and oriented in same direction)
– Also known as minisatellite sequences
– 10 to 100 bp in length
Variations of VNTR (D1S80)
allele lengths in 6 individuals
http://en.wikipedia.org/wiki/Variable_number_tandem_repeat/ Accessed: 6/14/10
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Types of Polymorphisms
• Short Tandem Repeat (STR)
– Occurs when a pattern of two or more nucleotides are repeated
– Repeated sequences are directly adjacent to each other
– Length ranges from 2 to 16 bp
– Also known as microsatellites
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STR Nomenclature
•
International Society for Forensic Genetics recommended nomenclature for STR loci in 1997
•
STRs within genes are designated according to gene name
– Examples:
• TH01 is in intron 1 of human tyrosine hydroxylase gene on chromosome 11
• TPOX is in intron 10 of human thyroid peroxidase gene on chromosome 2
•
Non‐gene associated STRs are designated by the D#S# system
– D stands for DNA, the following number designates the chromosome where STR is located
– S refers to a unique segment, followed by a number registered in the International Genome Database (GDB)
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Types of Polymorphisms
• Restriction Fragment Length Polymorphism (RFLP)
– A difference between 2 or more samples of homologous DNA molecules arising from differing locations of restriction sites
– Was the first DNA profiling technique used for gene mapping, human identification, and parentage testing
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Summary of Types of Polymorphisms
and Laboratory Methods
Polymorphism Structure
Detection Method
RFLP
One or more nucleotide changes
that affect the size of restriction
enzyme products
Southern blot
VNTR
Repeats of 10-50 base sequences Southern blot, PCR
in tandem
STR
Repeats of 1-10 base sequences
in tandem
PCR
SNP
Alterations of a single nucleotide
Sequencing, other
Buckingham L and Flaws, ML. Molecular Diagnostics: Fundamentals, Methods, & Clinical Applications. Philadelphia: F.A. Davis, 2007.
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RFLP Analysis
• RFLP analysis
– DNA sample is broken into pieces (digested) by restriction enzymes
– Resulting restriction fragments are separated according to lengths by gel electrophoresis and detected by Southern blot
– Fragment sizes may vary due to changes in nucleotide sequence in or between the recognition sites of a restriction enzyme
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RFLP Analysis
• A small segment of genome is being detected by a DNA probe (thicker line)
• In allele “A”, genome is cleaved by restriction enzyme at three nearby sites (triangles)
• Only rightmost fragment will be detected by probe
• In allele “a”, restriction site 2 has been lost by a mutation
• Probe now detects the larger fused fragment running from sites 1 to 3
http://en.wikipedia.org/wiki/Restriction_fragment_length_polymorphism. Accessed 6/15/2010.
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RFLP Analysis
• In this example, the probe and restriction enzyme are chosen to detect a region which includes a VNTR segment (boxes)
• In allele “c” there are 5 repeats in the VNTR and probe detects a longer fragment between the 2 restriction sites
• In allele “d” there are only 2 repeats in the VNTR, so probe detects a shorter fragment between the same 2 restriction sites
http://en.wikipedia.org/wiki/Restriction_fragment_length_polymorphism. Accessed 6/15/2010.
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RFLP and Parentage Testing
• A gene or region of DNA (locus) will have several versions (alleles) of the DNA sequence within that gene
• Each gene can have different alleles
• Different DNA sequences (alleles) can result in different traits, such as color
• Different DNA sequences (alleles) can have the same result in the expression of a gene
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DNA-based Parentage Testing
• Humans are diploid: each person has 2 alleles or copies of each locus
• Homozygous: alleles are the same
• Heterozygous: the two alleles are different
• More closely related individuals are likely to share more alleles than unrelated persons
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DNA-based Parentage Testing
• DNA is inherited as one chromosome complement from each parent
• Each chromosome carries its polymorphism
• Offspring inherits a combination of the parental polymorphisms
• Thus, through RFLP analysis, one can infer a parent’s contribution of alleles to a son or daughter from the combination of alleles in the child and those of the other parent
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RFLP and Parentage Testing
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Through RFLP analysis, the fragment
sizes of the child (electrophoresis lane 2)
is a combination of those from each parent
(father, lane 1) and (mother, lane 3)
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1
http://en.wikipedia.org/wiki/Polymerase_chain_reaction.
Accessed 6/15/2010.
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2
3
Electrophoresis of PCR-amplified DNA fragments.
(1) Father. (2) Child. (3) Mother. The child has inherited
some, but not all of the fingerprint of each of its parents,
giving it a new, unique fingerprint.
RFLP and Parentage Testing
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Animation
http://www.rvc.ac.uk/review/DNA_1/4_VNTRs.cfm
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RFLP and Parentage Testing
In a paternity test, alleles or fragment sizes of
offspring and mother are analyzed.
Remaining fragments (ones that do not match
mother) have to come from the father.
Alleged fathers are identified, or included,
based on the ability to provide remaining
alleles.
Aside from possible mutations, a difference in
just one allele may exclude paternity.
In this figure, only two loci are shown. Of the
alleged fathers (AF) shown, only one could
supply the fragments not supplied by the
mother.
Which is the alleged father?
Buckingham, L and Flaws, ML. Molecular Diagnostics: Fundamentals, Methods, & Clinical Applications. Philadelphia: F.A. Davis Company, 2007.
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Parentage Testing
• A parentage test requires analysis of at least eight loci.
• The more loci tested, the higher the probability of positive identification of the father.
• Paternity Index (PI):
– Calculated for each locus in which alleged father and child share an allele
– Is an expression of how many times more likely the child’s allele is inherited from alleged father than by random occurrence of the allele in the general population
– Allele occurring frequently in population has low PI; a rare allele has a high PI
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Other Methods for Detecting
Polymorphisms
• In contrast to VNTRs, smaller STRs can be amplified by PCR
–
–
–
–
Less specimen needed
Shorter time for analysis
Multiplexing and automation is facilitated
Can use fluorescence technology and capillary electrophoresis
• Reverse dot blot procedure: uses an array of immobilized probes to detect sequence polymorphisms
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Gender Identification
• Amelogenin gene
– Located on the X and Y chromosomes
– Not a STR
• Amelogenin locus
– Polymorphism located in second intron of amelogenin gene
– Y allele of gene is six bp larger in this region than X allele
– Amplification and electrophoretic resolution reveals 2 bands or peaks for males (XY) and one band or peak for females (XX)
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Amelogenin Locus
Males are heterozygous for the amelogenin locus (XY), and
females are homozygous for this locus (XX). Amplification of
amelogenin will produce a male-specific 218 bp product (Y allele)
in addition to the 212 bp product found on the X chromosome (X
allele).
Buckingham, L and Flaws, ML. Molecular Diagnostics: Fundamentals, Methods, & Clinical Applications.
Philadelphia: F.A. Davis Company, 2007.
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Y-STRs
• Y‐STRs are represented only once per genome and only in males
• A set of Y‐STR alleles comprises a haplotype, or series of linked alleles always inherited together, because Y chromosome cannot exchange information (recombine) with another Y chromosome.
• Marker alleles on Y chromosome are inherited from generation to generation in a single block.
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Y-STRs
• Less powerful than autosomal STR
• Useful where evidence consists of a mixture of male and female DNA (ex. rape cases)
• Used also in lineage studies involving paternally linked relationships and identification (ex. inheritance claims)
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Mitochondrial DNA
• Mitochondria contain a circular genome of 16,569 base pairs
• Structure is two strands of circular mtDNA
– Have an asymmetric distribution of Gs and Cs
– Generating a G‐rich heavy (H) strand and a C‐rich light (L) strand
• mtDNA are inherited maternally
• Used in forensic analysis (ex. open case files, missing persons cases, and typing ancient specimens)
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Identity Testing: Applications
• Applications
– Parentage
– Immigration
– Criminal investigation (forensics)
– Clinical
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Application: Immigration
The First Case, 1985
A young boy from Ghana leaves the U.K. to be reunited with his father in Ghana. He then returns to the U.K. To be reunited with his mother.
Was he the son or the nephew of this woman?
HLA techniques at that time were too crude to resolve this issue. Using minisatellite probes for hypervariable regions in human DNA, Alec Jeffreys indisputably showed that this was indeed the son and not the nephew of this woman. This immigration case led the way for DNA fingerprinting.
Tsongalis, GJ and Coleman, WB. Molecular Diagnostics: A Training and Study Guide. Washington, DC: American Association for Clinical
Chemistry (AACC) Press, 2002.
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Application: Forensics
The First Murder Case, 1986
Two schoolgirls in the quiet town of Leicester were murdered, one in 1983 and one in 1986. The circumstances of the rapes and murder were identical. So when a young man confessed, the authorities thought they had the assailant for both crimes. Semen swabs had been taken from both girls and DNA analysis was requested. The samples were from the same individual, but not the one who had confessed. Copyright 2010 American Society of Cytopathology
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Application: Forensics
The First Murder Case, 1986 (cont.)
A call was then put out to all local males between the ages of 17 and 34 to be subjected to DNA fingerprint analysis. The true murderer had a workmate go in his place. As the investigation dragged on, the proxy told a friend at the local pub that he had stood in for someone as part of the DNA test. A woman overheard the conversation and reported this to authorities. Tsongalis, GJ and Coleman, WB. Molecular Diagnostics: A Training and Study Guide. Washington, DC: American Association for
Clinical Chemistry (AACC) Press, 2002.
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Applications for Identity Testing in
Clinical Laboratory
•
•
•
•
•
Specimen identification
Urine donor identification (toxicology)
Pre‐employment drug testing
Confirmation for other civil cases
Monitoring bone marrow transplant chimerism
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Application: Specimen Identification
A 51‐year‐old man underwent core biopsy of the prostate to rule out carcinoma. Several H&E slides were received that showed a fragment of carcinomatous epithelium adjacent to benign prostate core samples. The origin of the neoplastic tissue was questioned. Copyright 2010 American Society of Cytopathology
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Specimen Identification Testing Results
Specimen
LDLR
GYPA
HBGG
D7S8
GC
TPO
THO
Blank
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-----
-----
-----
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Control
B,B
A,B
A,A
A,B
B,B
182/194
243
Prostate
B,B
A,B
A,B
A,A
C,C
230 bp
181 bp
? Tissue
A,B
A,B
A,B
A,A
A,C
230 bp
181/189 bp
The blank and control results indicated there were no problems
during the testing; however, since there are 2 loci (LDLR and GC)
that differ from the patient’s benign prostate tissue and the neoplastic
tissue, it is possible there was contamination during the tissue
processing.
Tsongalis, GJ and Coleman, WB. Molecular Diagnostics: A Training and Study Guide. Washington, DC:
American Association for Clinical Chemistry (AACC) Press, 2002.
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Application: Pre-employment Drug
Screening
A pre‐placement urine drug test conducted in a young man was positive for 480 ng/mL benzolylecognine. When contacted by the medical review officer, the donor denied using drugs and said the collector must have mixed up samples.
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Pre-employment Drug Screening Results
Specimen
DQA1
LDLR
GYPA
HBGG
D7S8
GC
Blank
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Control
1.1,4
B,B
A,B
A,A
A,B
B,B
Urine
1.1,1.2
A,A
A,B
B,B
A,A
B,B
Blood
1.1,1.2
A,A
A,B
B,B
A,A
B,B
The blank and control results indicated there were no
problems during testing; however, a blood sample taken
from the applicant matched the loci of the applicant’s urine
sample, thus confirming the urine sample was his.
Tsongalis, GJ and Coleman, WB. Molecular Diagnostics: A Training and Study Guide. Washington, DC: American
Association for Clinical Chemistry (AACC) Press, 2002.
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Bone Marrow Transplant Typing and
Monitoring
• Potential donors are tested for immunological compatibility
– Performed by analyzing HLA locus using sequence‐specific PCR or sequence‐based typing
• Engraftment: Donor cells reconstitute the recipient’s bone marrow
• Recipient is genetic chimera (recipient has body and blood cells of separate genetic origins)
• Engraftment monitoring by PCR amplification of STRs, capillary electrophoresis, and fluorescent detection
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