5162 G->A
Base position
RQ197G
Original aa
aa position
Replacement
Original base
Deletion: 197delAG
Insertion: 2552insT
Replacement
DNA sequence
Amino Acid Sequence
Type of
Mutation
ATG CAG GTG ACC TCA GTG
M
Q
V
T
S
V
none
ATG CAG GTT ACC TCA GTG
M
Q
V
T
S
V
silent
CAG CTG TCA GTG
M
Q
L
T
S
V
conservative
ATG CCG GTG ACC TCA GTG
M
P
V
T
S
V
nonconservative
ATG CAG GTG ACC TGA GTG
M
Q
V
T
ter
ATG CAG GTG AAC CTC AGT G
M
Q
V
N
L
nonsense
S
frameshift
conformers
1. Amplify region to be scanned using PCR.
Normal control
Test (with mutation)
2. Denature and dilute
the PCR products.
PCR products
3. Separate conformers by
PAGE or CGE.
Single strands
(conformers)
4. Analyze results by comparison to reference normal control (+).
PAGE
+
mut
CGE
+/mut
+
mut
+/mut
5. Detect PAGE bands by silver staining.
T1 T2 NC
T1: test sample without mutation
T2: test sample with mutation
NC: normal control
m/+ +/+ m/m
m/+ +/+ m/m
+ probe
m probe
1
2
m
+ probe
+
N
1
2
m
m probe
+
N
Tm
• Ethidium bromide
• SyBrGreen
FRET
loss of
fluorescence
Fluorescence
%SS
DS=SS
Tm
%DS
50
Temperature (°C)
80
characteristic Tm
Heterozygous (m/+)
%SS
DS=SS
Homozygous
mutant (m/m)
Homozygous
normal (+/+)
%DS
50
Temperature (°C)
80
Df/Dt
Normal
Heterozygous
mutant
Temperature (°C)
Mutant Tm
Normal Tm
• Reverse dot blot
Method
Substrate
Detection
macroarray
nitrocellulose
radioactive,
chemiluminescent,
chromogenic
microarray
glass, nitrocellulose on
glass
fluorescent
high-density
oligonucleotide arrays
glass
fluorescent
microelectronic arrays
electrode grid
fluorescent
Method
Array
Application
comparative genomic
hybridization (CGH)
microarray,
macroarray
detection of genomic
amplifications and
deletions
expression array
microarray,
macroarray
detection of relative
changes in gene
expression
SNP detection,
mutation analysis,
sequencing
high-density
oligonucleotide array
detection of singlebase differences in
DNA
C A T A T
A G C T G
T T C C G
(10–25mers)
• Tiling
array
readers
Results displayed in graphical form.
Normal sequence
(TCG)
Heterozygous
(TCG>TAG)
A C G T del
A C G T del
A C G T del
A C G T del
A C G T del
A C G T del
Represents five probes, each carrying
the indicated base or deletion at the
same position
SSP-PCR
Primer
G
C
Normal template
G
Mutant template
(Amplification)
(No amplification)
T
(1) GAAGTTGCATTTTATAAACCTT->
AAAATGAAGTTGTCATTTTATAAACCTTTTAAAAAGATATATATATA
TGTTTTTTCTAATGTGTTAAAGTTCATTGGAACAGAAAGAAATGGAT
TTATGTGCTGTTCGCGTTGAAGAAGTACAAAAT
(2) ATTAATGCTATGCAGAAAATGTTAGAG-> (only in normal)
GTCATTAATGCTATGCAGAAAATGTTAG[AG]TGTCCCATCTGGTAA
(only in mutant)<--ATC - - ACAGGGTAGACCATT
GTGAGCACAAGAGTGTATTAATTTGGGATTCCTATGATTATCTCCTA
CAGT (3)
TGCAAATGAACAGAATTGACCTTACATACTA
<- CTTGTCTT AACTGGAATGTAT (4)
+mm+
230 bp (1) and (4)
180 bp (1) and (3)
120 bp (2) and (4)
Product
of primers
(1) and (3)
is specific for
mutation.
Normal probe (FAM)
Mutant probe (VIC)
Normal
Green signal
Mutant
Red signal
Mutant allele
(VIC)
Het
Mut
NL
Normal allele (FAM)
NL: … GTCA GGGTCC
Mut: … GTCA GGATCC
NL
Mut
U
Agarose
gel:
U: uncut
C: cut
C
U
C
GTGC…
CTGC…
Het
U
C
Mutation
Mix, denature
Renature
Homoduplexes
Heteroduplexes
Homoduplexes
not cleaved by enzyme
M
NL
Heteroduplexes
cleaved by enzyme
Mutants
Cleaved
fragments
indicate presence
of mutation.
mut probe
A
wt probe
G
T
Cleavage
A
T
(No cleavage)
Complex formation
F
Q
A
Cleavage
F
Detection