Download methods of Screening3

Mutation Screening
Dr. Derakhshandeh, PhD
Quantitative PCR
and Dosage
2
Quantitative PCR and Dosage
used in a number of
diagnostic applications eg.
 SMA
 trisomy 21
 X-linked
Agammaglobulinemia
3
SMA
4
CLASSIFICATION
SMA TYPE I (WerdnigHoffmann)
SMA TYPE II (Classic)
SMA TYPE III
(Kugelberg-Welander)
5
SMA TYPE I
Severe form of SMA
Onset : first 6 months
Death : < 2 year
Never raising the head
or sitting
6
SMA TYPE I
7
SMA TYPE II
Less severe
Clinical appearing : < 18 months
Able to sit un aid
Death : about 9 years
8
SMA TYPE III
Mildest form of SMA
Onset : > 18 months
Walking without aid
9
GENETIC MAP
Three candidate genes named SMN
(Survival Motor Neuron), NAIP (Neuronal
Apoptosis Inhibitory Protein) and P44 were
identified in this locus
Up to 95% of SMA patients (SMNI-III) are
homozygously deleted for two exons (7&8)
of both telomeric copy of SMN gene (SMNt)
10
Deletions
 Up to 5% of SMA patients have frameshift
mutations, gene conversions and point
mutation
Exons 5 and 6 of NAIPt gene are deleted
in approximately 50% of type I SMA and
18% of types II and III SMA
P44t is lacked or intrrupted in 73% of SMA
type I patients and 7% in types II and III
11
The summery of normal alleles (N) , mutant alleles (M)
and deletion types (D) of SMN
12
MOLECULAR DIAGNOSIS & PND
PCR-SSCP or PCR-RFLP of SMN gene
enables confirmation of a suspected
clinical diagnosis of SMA or prenatal
diagnosis
These two techniques based on
nucleotide differences of both exon 7 and
exon 8 of telomeric and centromeric
copy of SMN
13
14
15
16
17
18
Deletion Analysis of SMN gene
• Exones 7 and 8 of SMN
gene were amplified
and cut by Dra I and
Dde I , respectively.
(only centromeric
copy is cutted)
• Absence of SMNt
exone(s) 7 (and 8)
confirm diagnosis of
SMA
19
20
Exon 7, DraI
188 bp
164 bp
Exon 8, DdeI
188 bp
123 bp
65 bp
21
SMN Deletion Analysis
Derakhshande et al.
(Farhud Genetic Lab/ NRCGEB)
• SMNt Exon 7 is
deleted in
affected child
22
NAIP Gene Deletion Analysis
• Exones 5 & 6 of NAIP gene were amplified with exon
13 which was the internal control
• Absence of exon
5 and exon 6 ( which
only exist within the
telomeric functional
copy of NAIP) was
detected in ~50% of
type I SMA and 18%
of types II and III SMA
23
NAIP Deletion analysis
Derakhshande et al.
(Farhud Genetic Lab/ NRCGEB)
24
25
• The objective of this study was to genetically
characterize the childhood onset spinal muscular
atrophy in Iran.
SMN
NAIP
Deletion of exon 7 & 8
Deletion of exon 5 & 6
SMA type I (n=70)
70(100%)
61(87%)
SMA type II (n=3)
2(66%)
1(33%)
SMA type III (n=2)
1(50%)
0(0%)
26
• Various deletion haplotypes were constructed by using
genotypes of SMN and NAIP genes.
• Haplotype A, which has the deletions of all two involved genes,
were deleted in approximately 83% of type I and II SMA but not
in type III and was found predominantly in the severe group
with an early onset at less than 6 month of age.
• we report Thirty four our experiences for prenatal diagnosis
Haplotypes (%)
A
B
C
SMA type I
(n=70)
87
100
0
SMA type II
(n=3)
33
66
33
SMA type III
(n=2)
0
50
50
27
• These studies suggested that the frequency of
gene deletions of SMN1 and NAIP gene is a
few higher than previous reports. It is may be
due to high rate of consanguine marriage by
Iranian Muslims (96 % in this families). Thus, the
conformation of SMA related gene deletion will
also be a useful tool for the pre and postnatal
diagnostic. In addition to common PCR
methods for SMN exon 7 and 8 and NAIP exons
4 and 5, we also conducted multiplex PCR of
exon 5, 6 and 13 of the NAIP telomere in one
reaction.
28
Quantitative PCR and Dosage
• Products can be measured by image
scanning of agarose gels or Polaroid
pictures
• Method is not sensitive to template
concentration (1.3g to 2.7pg DNA used)
but can be affected by template
quality so preparation is important.
29
More complex method
SMA
This method allows
quantitation of the copy
number of SMNT and SMNC
genes on chromosome 5q12
30
The homologous gene quantitative
polymerase chain reaction(HGQPCR)
31
Detection of trisomy 21 by HGQ-PCR
Lanes 1–16 Serial dilutions for a normal individual and
aDown’s syndrome patient used to evaluate the optimal
HGQPCR
32
Sequencing
33
Top and bottom strand
differences for a point mutation
34
Top and bottom strand differences for
a heterozygous base
(Click for full size image)
Figure 2. Top and bottom strand differences for a heterozygous base
35
Sequencing
36
Forensics
37
Forensics
PCR analysis PCR
(polymerase chain
reaction)
RFLP analysis
STR analysis Short tandem
repeat (STR) technology is
a forensic analysis
For example, the likelihood
that any two individuals
(except identical twins) will
have the same 13-loci DNA
profile can be as high as 1
in 1 billion or greater.
38
Forensics
Mitochondrial DNA Analysis
Mitochondrial DNA analysis
(mtDNA) can be used to examine
the DNA from samples that
cannot be analyzed by RFLP or
STR
Nuclear DNA must be extracted
from samples for use in RFLP, PCR,
and STR
mtDNA analysis uses DNA
extracted from another cellular
organelle called a mitochondrion
39
Forensics
While older biological samples that
lack nucleated cellular material, such
as hair, bones, and teeth, cannot be
analyzed with STR and RFLP
they can be analyzed with mtDNA
In the investigation of cases that have
gone unsolved for many years, mtDNA
is extremely valuable.
40
Forensics
Y-Chromosome Analysis
The Y chromosome is passed directly from
father to son
so the analysis of genetic markers on the
Y chromosome is especially useful for
tracing relationships among males
this technique can be very valuable if the
laboratory detects complex mixtures
(multiple male contributors)
41
Reverse Dot Blot for
Human Mutation
Detection
Introduction
• Reverse dot blot (RDB)
• or reverse allele specific
oligonucleotide (Reverse
ASO)
• hybridization
• important method for
genotyping common
human mutations
43
Commonly used in:
• a high mutation spectrum
• high frequency disorders
such as:
–
–
–
–
–
cystic fibrosis
hemoglobin C (HbC)
hemoglobin E (HbE)
hemoglobin S (HbS)
ß-thalassemias
44
Location of mutations in the b-globin gene
45
RDB procedure
• exons (or other regions of
interest)
• amplified by the
polymerase chain reaction
(PCR)
• using labeled
oligonucleotide primers
• 5' biotin label on PCR
primers
46
Amplicons
• Amplification products
• denatured
• hybridized
– with mutation specific DNA
probes
• covalently bound to solid
membran
47
Incubation
• nucleic acids: incubated
with an enzyme
conjugated to streptavidin.
• enzyme-conjugated,
streptavidin-biotin-nucleic
acid complex is then
washed
• incubated with
– a chromogenic
– or luminogenic substrate,
which allows visualization of
hybridized spots
48
Materials and Methods
• Total genomic DNA
• extracted from peripheral
blood leukocytes
• Amniotic fluid cells (AF)
• chorionic villi (CVS)
49
A woman having amniocentesis
50
Oligonucleotide probes
• A C6-amino-link phosphoramidite
• amino moiety on the 5' end of the
product
51
Oligonucleotides used for reverse dot blot (RDB)
52
RDB
53
Reverse dot (RDB) blot hybridization for detection of
10 common β-thalassaemia mutations
54
b-thalassemia Patients
55
Screening for causal mutations
• genomic DNA from
patient blood samples
• reverse dot blot (RDB)
• amplification refractory
mutation systempolymerase chain
reaction (ARMSPCR)
• DNA sequencing
56
PCR from genomic DNA
720 bp
57
58
59
Strips
1
2
NM
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
60
The Blots
61
Comparison of different factors determining the efficiency of ARMS and
reverse hybridization in beta thalassemia diagnosis
ARMS
Reverse hybridization
Turnover time
several days
6-8 hours
Equipment
Expensive (large PCR
machine, gel
electrophoresis,
photodocumentationsyst
em
Less expensive (small PCR
machine, agarose gel,
small shaking water bath)
Number of PCR reactions
per sample
8-88
1
Documentation
Requires documentation
process after experiment
Self-documented
Technician time (number
of patients: time in days)
1:1
10:1
Starting material
Depending on the
number of PCR reactions
0.5 μg genomic DNA for
just one PCR reaction
Toxic materials
Ethidium bromide
(carcinogen)
None
62
In the late stages of muscular dystrophy, fat and
connective tissue often replace muscle fibers.
63
64
DMD
65
Orthopaedic management of
patients with Duchenne's muscular
dystrophy
66
L
A
B
C
D
E
F
G
H
L
~95% of deletions can be detected in males using multiplex PCR
67
MAPH
• Detection of deletions/duplication
mutations in Duchenne Muscular
Dystrophy using: Multiplex
Amplifiable Probe Hybridisation
(MAPH)
68
MAPH
• Although ~95% of deletions can be detected in
males using multiplex PCR
• other methods must be used to determine
duplications, as well as the carrier status of
females
• The most commonly applied methods are
quantitative multiplex PCR and quantitative
Southern blotting
• The drawback of quantitative multiplex PCR is
that often not all mutations are examined
• meaning that small and rare mutations are
missed
69
MAPH
• Using high-quality Southern blots it is possible to
perform a quantitative analysis and detect
duplications
• this technique is time consuming
• it is difficult to exactly determine the duplication
• it can be difficult to detect duplications in
females and triplications will be missed
Armour et al (Nucl.Acids Res. 2000)
70
• system for analysing all 79
exons of the DMD gene for
deletions and duplications
• MAPH is based on a
quantitative PCR of short
DNA probes recovered
after hybridisation to
immobilized genomic DNA
71
• 1 ug of denatured genomic DNA is spotted
on a small nylon filter
• hybridized overnight in a solution containing
one of the probe mixes
• Following stringent washing the next day
the filter is placed in a PCR tube
• and a short PCR reaction is performed
• This releases the specifically-bound probes
into the solution
• An aliquot of this is transferred to a second,
quantitative PCR reaction
72