BTN Protocol Guide-08

| Microbial Identiﬁ cation
Comparative Sequence Analysis
Sequenom, Inc.
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San Diego, CA 92121
3595 John Hopkins Ct
San Diego, CA 92121
aloria@sequenom.com
www.sequenom.com
www.sequenom.com
Introducing the new MassARRAY
®
858-202-9194
Solution for The three step protocol is PCR based and allows for
Molecular Typing the amplification of a target region of interest.
Following SAP treatment, the samples are subject to
The MassARRAY Comparative Sequence Analysis in vitro transcription and enzymatic base - specific
Introducing the New MassARRA
application sets a new precedence
Y
in
®
m
Solution for Molecular
olecular cleavage. After sample conditioning, the cleavage
typing. Recently, a new and optimized protocol has mixture is dispensed onto aT 3yping84 well SpectroChip
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.
been developed based on the MassCLEAVE
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The SpectroChip is introduced into the MassARRAY
chemistry to enable accurate comparative sequence Compact
TM
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The MassARRAY Compara
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Sequence
he applica
Analysis
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a

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nce
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a
ences
Following
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e
the
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are
DI-TO
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ss
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new precedent in molecular
as a comp
typing.
arative
Recently
measure
, a new
to
and
identify
optimized
microbes,
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and
etry.
enzymatic base-speciﬁ c cleavage. After sample
protocol has been developed
viruses, o
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tide.
The application SpectroChip
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.
e
The
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SpectroChip
lysis softwa
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A graphic
into
al s
the
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Y
uses reference sequences The new as Maa sscomparaCLEAVEtive assmeasure ay is baseto d oidentify n an in vitroCompactinter
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fa cAnalyzer e of theand ID the resculealts vaage nd products mass spare ectraautoma is tically
microbes, viruses, or
tra
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nscrip
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nuclease e
with
nzym
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atic cleav
do
ag
wn
e of 500
analyzed by MALDI-TOF mass spectrometr
provided in Figure 2. The Compara
y.
t

ive Sequence
to a single nucleotide.
– 800 bp PCR products from the target region of Analysis software automatically generates a report
interest. A protocol overview is provided in Figure 1. containing ID results listing the best matching
Data
r
analysis
eference
is
s
performed
equences
on
fo
the
r e
Compara
ach targ
tive
et
Sequence
region of
Analysis
The new MassCLEAVE assay is based on an in vitro transcription software.inter eA stgra, aphical s well software as, sequinterface ence vaof riathe tionsID aresults nd newand mass
ribonuclease enzymatic cleavage of 500–800 bp PCR products spectra is provided in F
sequences.
igure 2.
from the target region of interest. A protocol overview is provided
in Figure 1.
genomic DNA
PCR and T7
SAP Treatment
SP6
MassCLEAVE
Assay
Results:
� ID
T7
In vitro Transcription
SP6SP6
� Sequence Variations
with T7 and SP6
T7 SP6
Polymerases � Clustering
U C U C
Figure
Figure
2. Dat
2.
a a
Dat
naly
a
s
anal
is so
ysi
ftw
s
ar
software
e interfac
inte
e fo
rfa
r C
c
o
e
m
for
par
Com
ative
parative
Sequence Analysis
Base-specific
RNA Cleavage Sequence Analysis
Figure 1: Overview of MassCLEAVE Assay
The Comparative Sequence Analysis software automatically
References:
Data Acquisition
The MassCLEAVE process is homogeneous and
generates a report containing ID results listing the best matching
• Honisch, C., et al. (2007). "Automated comparative
does not
T
requir
C
e purif
“G”
ication
“A
o
”
f the PCR product or reference
sequen
sequences
ce analysis
for
by
each
base
target
specific
region
cleavage
of interest,
and ma
as
ss
well as
subsequent analytes. The process is automatable
sequence varia
spectrome
tions and new sequences.
try for nucleic acid-based microbial typing.“
and thus amen
Sequence Compariso
able to high
n
throughput. Proc Natl Acad Sci USA 104(25): 10649 54.
Identification
Base-specific
Sequence V
clea
ariation Detectio
vage g
n
enerates a defined
References:• Herring, C. D., et al. (2006). "Comparative genome
experimental mass signal pattern of the sample. For
1. Honi
seq
sch
uen
, C.,
cing
et a

l.
of
(20
Esche
07). “
r
A
ichi
uto
a
ma
coli
ted c
allows
ompara
ob
tive
se
s
rvation
equenc

e
of
analysis
Figure 1: a Onavelyrvsiiesw , otf hMe aessxCpLeErAimVE enAtsasla ypattern is subsequently
by base
bacte
speci
rial
fic

c
evol
leav
ution
age an
o
d
n
ma
a lab
ss
o
s
rato
pect
ry
rom
time
etr
scale."
y for nuc
Nat
leic
Ge
aci
net
d-based
compared to an in silico reference mass signal
38(12): 1406-12.
The MassCLEAVE process
pattern d
is
er
homogeneous
ived from an in
and
divid
does
ual or
not
set
require
of reference
microbial typing.” Proc Natl Acad Sci USA 104(25): 10649-54.
puriﬁ cation of the PCR seqproduct uences. or Dsubsequent ifferences banalytes.etween t The he eprocess xpected and
• Lefmann, M., et al. (2004). "Novel mass spectrometry-
is automatable and thus amenable to high throughput.
the observed mass signal pattern are interpreted and
2. Herr
based
ing, C.
tool
D., e
fo
t a
r
l.
ge
(2
notypic
006). “C
identif
ompa
icatio
rative
n
ge
of
no
mycob
me seq
a
u
cte
enc
ria."
ing of
enable identification of sequence variations.
Escheric
J
hi
Clin
a co
Mi
li
crobi
allow
ol
s o
42(1
bser
)
v
:
a
339
tion
-46.
of bacterial evolution on a laboratory
timescale.” Nat Genet 38(12): 1406-12.
Base-speciﬁ c cleavage generates a deﬁ ned experimental mass
signal pattern of the sample. For analysis, the experimental pattern
3. Lefmann, M., et al. (2004). “Novel mass spectrometry-based tool for
is subsequently compared to an in silico reference mass signal
genotypic identification of mycobacteria.” J Clin Microbiol 42(1): 339-46.
pattern derived from an individual or set of reference sequences.
Differences between the expected and the observed mass signal
pattern are interpreted and enable identiﬁ cation of sequence
variations.
The three step protocol is PCR based and allows for the ampliﬁ cation
of a target region of interest.
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