Transcript 11 Molecular Diagnostics

Molecular Diagnostics
DNA Polymorphisms and Human
Identification
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Molecular Diagnostics
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Polymorphism
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A DNA polymorphism is a sequence
difference compared to a reference standard
that is present in at least 1–2% of a
population.
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Polymorphisms can be single bases or
thousands of bases.
Polymorphisms may or may not have phenotypic
effects.
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Molecular Diagnostics
Polymorphic DNA Sequences
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Polymorphisms are found throughout the
genome.
If the location of a polymorphic sequence is
known, it can serve as a landmark or marker
for locating other genes or genetics regions.
Each polymorphic marker has different
versions or alleles.
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Molecular Diagnostics
Types of Useful Polymorphisms
and Laboratory Methods
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Molecular Diagnostics
RFLP Typing
Restriction fragment
sizes are altered by
changes in or
between enzyme
recognition sites.
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Molecular Diagnostics
RFLP Typing
The presence of
RFLP is inferred
from changes in
fragment sizes.
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Molecular Diagnostics
RFLP Typing
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Molecular Diagnostics
RFLP and Parentage Testing
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RFLP genotypes are inherited.
For each locus, one allele is inherited from each
parent.
Southern blot
band patterns
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Molecular Diagnostics
RFLP and Parentage Testing
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Who is the alleged
father?
Of the two alleged
fathers shown, only one
could supply the
fragments not supplied
by the mother.
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Molecular Diagnostics
Evidence Testing by RFLP
Which suspect—S1 or S2—was at the crime scene?
(V = victim, E = crime scene evidence, M = molecular weight
standard)
M S1 S2 V E M
M S1 S2 V E M
M S1 S2 V E M
Locus 1
Locus 2
Locus 3
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Molecular Diagnostics
Short Tandem Repeat
Polymorphisms (STR)
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STR are repeats of nucleotide sequences.
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AAAAAA… - mononucleotide
ATATAT… - dinucleotide
TAGTAGTAG… - trinucleotide
TAGTTAGTTAGT… - tetranucleotide
TAGGCTAGGCTAGGC… - pentanucleotide
Different alleles contain different numbers of
repeats.
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TTCTTCTTCTTC - four repeat allele
TTCTTCTTCTTCTTC - five repeat allele
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Molecular Diagnostics
Short Tandem Repeat
Polymorphisms
STR alleles can be analyzed by fragment size
(Southern blot).
One repeat unit
Restriction site
Allele 1
GTTCTAGCGGCCGTGGCAGCTAGCTAGCTAGCT GCTGGGCCGTGG
CAAGATCG CCGGCACCG TCGATCGATCGATCGA CGACCCGGCACC
tandem repeat
Allele 2
GTTCTAGCGGCCGTGGCAGCTAGCTAGCT GCTGGGCCGTGG
CAAGATCG CCGGCACCG TCGATCGATCGA CGACCCGGCACC
Allele
M 1 2 M
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Molecular Diagnostics
Short Tandem Repeat
Polymorphisms
STR alleles can also be analyzed by amplicon size
(PCR).
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Molecular Diagnostics
Short Tandem Repeat
Polymorphisms
Allelic ladders are standards representing all
alleles observed in a population.
11 repeats
(Allelic ladder)
5 repeats
Genotype: 7,9
Genotype: 6,8
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Molecular Diagnostics
Short Tandem Repeat
Polymorphisms
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Multiple loci are genotyped in the same reaction
using multiplex PCR.
Allelic ladders must not overlap in the same
reaction.
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Molecular Diagnostics
Short Tandem Repeat
Polymorphisms by Multiplex PCR
FGA
PentaE
TPOX
D18S51
D2S11
D8S1179
THO1
vWA
D3S1358
STR Nomenclature
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The International Society for Forensic Genetics
recommended nomenclature for STR loci in 1997:
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STRs within genes are designated according to the gene name
(no phenotypic effect with respect to these genes):
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TH01 is in intron 1 of the human tyrosine hydroxylase gene on
chromosome 11
Non–gene associated STRs are designated by the D#S#
system:
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D stands for DNA
The following number designates the chromosome where the STR
is located (1-22, X or Y).
S refers to a unique segment, followed by a number registered in
the International Genome Database (GDB).
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Molecular Diagnostics
Gender Identification:
Amelogenin Locus, HUMAMEL
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The amelogenin locus is not an STR (but along with
STR).
The HUMAMEL gene codes for amelogenin-like
protein.
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located on the X (Xp22.1–22.3) and Y chromosomes
required for embryonic development and tooth maturation
Polymorphism is located in the second intron of the
amelogenin gene:
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X allele = 212 bp
Y allele = 218 bp (longer)
Males (X, Y)
Heterozygous (2 bands)
Females (X, X)
Homozygous (1 band)
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Molecular Diagnostics
Analysis of STR PCR Test Results:
STR genotypes are analyzed using gel or
capillary gel electrophoresis.
11 repeats
11 repeats
5 repeats
5 repeats
(Allelic ladder)
Genotype: 7,9
Identity testing by STR-PCR
A matching genotype is not necessarily an absolute
determination of identity of an individual.
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Genetic concordance: is a
term used where all locus
genotypes (alleles) from two
sources are the same.
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Concordance is interpreted as
inclusion of a single individual
as the donor of both
genotypes.
Two samples are considered
different if at least one locus
A microvariant allele (15.2) migrates
genotype differs (exclusion).
between the full-length alleles
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Molecular Diagnostics
Parentage Testing by STR-PCR
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Which alleged father’s
Child and parent are not
necessarily in complete Genetic genotype has the
paternal alleles?
concordance
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Mutational events may generate a
new allele in the offspring, and this
difference may not rule out paternity.
Child
Mother
Father
Locus
D3S1358
vWA
FGA
TH01
TPOX
CSF1PO
D5S818
D13S317
Child
16/17
14/18
21/24
6
10/11
11/12
11/13
9/12
Mother
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16/18
20/21
6/9.3
10/11
12
10/11
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1
17
14/15
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6/9
8/11
11
13
12/13
2
17
16/17
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6/7
8/9
11/13
9/13
11/12
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Molecular Diagnostics
Short Tandem Repeat Polymorphisms:
Y-STR
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The Y chromosome is inherited in a block without
recombination (Y chromosome cannot exchange
information)
STR on the Y chromosome are inherited paternally as a
haplotype.
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Series of linked alleles always inherited together
Thus, marker alleles on the Y chromosome are inherited from
generation to generation in a single block.
Y haplotypes are used for exclusion and paternal
lineage analysis.
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Except for rare mutation events, every male member of a
family (brothers, uncles, cousins, and grandparents) will have
the same Y-chromosome haplotype.
Engraftment Testing Using DNA
Polymorphisms
Recipient receives
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Allogeneic donor and recipient
immune compatibility is tested
prior to the transplant by HLA
typing.
Sequence polymorphisms
(alleles) in the HLA locus are
compared with those of the
recipient to determine which
donor would be most tolerated by
the recipient immune system.
Donors may be known or related
to the patient or anonymous
unrelated contributors (matched
unrelated donor).
his or her own
purged cells
Recipient
receives donor
cells
A recipient with donor marrow is a chimera.
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Molecular Diagnostics
Chimerism Testing Using STR
There are two parts to chimerism testing:
pretransplant informative analysis and posttransplant engraftment analysis
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donor cells can be monitored by following donor
polymorphisms in the recipient blood and bone marrow
Before transplant
Donor
Recipient
After transplant
Complete (Full)
Mixed
Graft failure
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Molecular Diagnostics
Chimerism Testing Using STR:
Informative Analysis
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STR are scanned to find informative loci (donor alleles
differ from recipient alleles).
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Noninformative loci: the donor and the recipient have the
same alleles.
Donor-informative loci: donor and recipient share one allele,
and the donor has a unique allele.
Recipient informative loci: the unique allele is in the recipient
Full chimerism: only the donor alleles are detected in
the recipient
Mixed chimerism: a mixture of donor and recipient
alleles are present
Graft failure: only recipient alleles are detectable
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Molecular Diagnostics
Which loci are informative?
GF: graft failure
MC: mixed chimerism
FC: full chimerism
vWA
TH01
Amel
TPOX
CSF1PO
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Molecular Diagnostics
Chimerism Testing Using STR:
Engraftment Analysis
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Using informative loci, peak areas are determined in
fluorescence units or from densitometry scans of gel
bands.
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A(R) = area under recipient-specific peaks
A(D) = area under donor-specific peaks
A(R) + A(D)
A(D)
A(R)
% Recipient DNA =
A(R)
A(R) + A(D)
× 100
% Recipient DNA =
A(R)
A(R) + A(D)
× 100
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Molecular Diagnostics
Chimerism Analysis of Cellular
Subsets
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Cell subsets (T cells, granulocytes, NK cells,
etc.) engraft with different kinetics.
Analysis of cellular subsets provides a more
detailed description of the engrafting cell
population.
Analysis of cellular subsets also increases the
sensitivity of the engraftment assay.
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Molecular Diagnostics
Chimerism Analysis of Cellular
Subsets
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T cells (CD3), NK cells (CD56), granulocytes,
myeloid cells (CD13, CD33), myelomonocytic
cells (CD14), B cells (CD19), stem cells (CD34)
Methods
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Flow cytometric sorting
Immunomagnetic cell sorting
Immunohistochemistry + XY FISH
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Molecular Diagnostics
Chimerism Analysis of Cellular
Subsets
Detection of different levels of engraftment in
cellular subsets is split chimerism.
R
D
T
R = Recipient alleles
D = Donor alleles
T = T-cell subset (mostly recipient)
G = Granulocyte subset (mostly donor)
G
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Molecular Diagnostics
Single Nucleotide Polymorphisms
(SNP)
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Single-nucleotide differences between DNA
sequences.
One SNP occurs approximately every 1,250
base pairs in human DNA.
SNPs are detected by sequencing, melt curve
analysis, or other methods.
99% have no biological effect;
60,000 are within genes.
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Molecular Diagnostics
SNP Detection by Sequencing
T/T
5′ AGTCTG
T/A
5′ AG(T/A)CTG
A/A
5′ AGACTG
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Molecular Diagnostics
SNP Haplotypes
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SNPs are inherited in blocks
or haplotypes.
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Sections of DNA along
chromosomes can be inherited
as a unit or block of sequence
No recombination occurs within
the block.
All the SNPs on that block
comprise a haplotype.
SNPs can be used for
mapping genes, human
identification, chimerism
analysis, and many other
applications.