hma & hta: theoretical and practical issues. topics development of the ( env ) hma strategies...
TRANSCRIPT
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HMA & HTA:Theoretical and Practical Issues
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Topics
Development of the (env) HMA Strategies for subtype determination
Heteroduplex Tracking Assay
Importance of PCR template
quantitation
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Heteroduplex Mobility Assay (HMA)
TAA
TTA
AATTTA
Denature,Reanneal
Denature,Reanneal
AcrylamideGel
Homoduplexes
Heteroduplexes
AcrylamideGel Heteroduplexes
Homoduplexes
T CA G
C T G A
G A
T C
A G
C T
G CA TG C
A T
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Heat
Ureaor
More Heator Urea
Melt &Reanneal
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Detection of heteroduplexes
25--
50035-+
50035+-
50035-+
5003535 3535-- ---- --
500100501035Cycles-Resolve-Heat/Cool
5DNA (ng)
Agarose gel
Acrylamide gel
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M 1.3 1.4 1.8 3.7 3.9 4.0 4.2 4.4 4.7 4.9 M
Detection of mismatches (without Indels)
% mismatches
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MW215X201215X202215X204215X207215X210215X216
16X21416X21516X21616X20116X20216X204
9X23 5X16 224X165X212 5X2145X215
MW0.18%
0.36%0.9% 1.08% 1.45%
2.0%
EACH BAR INDICATES THE POSITION OF NUCLEOTIDE SUBSTITUTIONS BETWEEN THE TWO SEQUENCES
THE POSITION OF THE SUBSTITUTION IS INDICATED ON THE RIGHT5% polyacrylamide, 200 volts, 3 hours.
676 bp
517 bp
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1 101 201 301 401 501 601 701 801 901 1001 1101 1201 1301 1401
ED5-ED12ES7-ES8
ED31-ED33
V1-V2 V3loop V4 V5
Curves: Gaps
Gaps +Base changes
PCR Fragments Used For HIV-1 env Heteroduplex Tracking
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Spectrum of Gel Shifts (env HMA)
Within an individual
Between individuals (same subtype)
Between individuals(different subtype)
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% Divergence
0
0.25
0.5
0.75
10 5 10 15 20 25
US- AFRUS- USInt ras ubje c tNo Gaps
M M
Gel Shift versus Divergence (V3-V5 Region)
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0.16 0.95 1.42 0.79 0.95 1.11- - - + + +
M
M3 Seq.
Impact of INDELS* on
heteroduplex mobility
*INDELS - Insertions or deletions # of bands =(# of Distinguishable Species)2 - (# of D.S.)
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vif
rev
env
rev
pol
gag
1000 2000 3000 4000 5000 6000 7000 8000 9000
nef
vpr
•
•tat
•
•vpu
•tat
•
HIV-1 Env Gene Amplimer SitesED5 ED12
ES7ED31 ED33
ES8
V3 Loop V4 V5
gp120
V1-V2
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BetweenM & O groups
Withina person or
subtype
5 300
20
40
60
80
100
120
10 15 20 25 35 40 45 50
Within or betweensubtypes
1.2kb ED5-ED12
DNA divergence in env regions
% DNA Distance
0.5kb ED31-33
0.63kb ES7-ES8
~Range of HMA in neutral 5% Acryl. gels
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M Ho A B C D E F
Importance of Multiple Comparisons
He
*Please avoid “sub-subtype” designationsbased on affinity to different references*
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He
Ho A B C D E F Ho A B C D E F
93RW003 93RW004
Highly Divergent Subtypes
ssDNA
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M Ho A B C D E F
4vs6
RU103 vs:
Identification of a new subtype
He
Ho
He
ss
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M HoB D E
He
He
Ho
93TH063HoB D E
93TH064HoB D E
93TH062
Efficient Subtype Analysis
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M HoB D E
He
He
Ho
94TH135HoB D E
94TH1364HoB D E
94TH137
Importance of the “Ho” Control
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Vs.TH239
B
Vs.TH129
E
vs. TH239B
vs. TH129E
Rapid Subtyping in Thailand
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1.2kb FRAGMENTS (ED5/ED12)
M
A1
A3+
A2
A3+
B1
B3+
B2
B1+
C1
C3+
C2+
C3
D1
D2+
D3
D1+
E1
E2+
E3
E1+
F1
F2+
G1
G2+
G3
G2+
A1
A2+
B2
B3+
C1
C2+
D2+
D3
E2
E3+
G1
G3+
C1+
C4
C2+
C4
C3
C4+
WITHIN SUBTYPE COMPARISONS
M A2 B1 C2 D1 E2 F2 G2 H2 B2
B2 + OTHER SUBTYPES
Inter- and Intra-Subtype Comparisons
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M A2 B1 C2 D1 E2 F2 G2 H2 B2
0.7kb FRAGMENTS (ES7/ES8)
M
A1
A3+
A2
A3+
B1
B3+
B2
B1+
C1
C3+
C2+
C3
D1
D2+
D3
D1+
E1
E2+
E3
E1+
F1
F2+
G1
G2+
G3
G2+
A1
A2+
B2
B3+
C1
C2+
D2+
D3
E2
E3+
G1
G3+
C1+
C4
C2+
C4
C3
C4+
WITHIN SUBTYPE COMPARISONSB2 + OTHER SUBTYPES
Inter- and Intra-Subtype Comparisons
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M A2 B1 C2 D1 E2 F2 G2 H2 B2
0.5kb FRAGMENTS (ED31/ED33)
M
A1
A3
+A2
A3
+B1
B3
+
B2
B1+
C1
C3
+C2+
C3
D1
D2
+
D3
D1+
E1
E2
+
E3
E1+
F1
F2
+G1
G2
+
G3
G2+
A1
A2
+B2
B3
+C1
C2
+D2+
D3
E2
E3
+G1
G3
+C1+
C4
C2+
C4
C3
C4+
WITHIN SUBTYPE COMPARISONSB2 + OTHER SUBTYPES
Inter- and Intra-Subtype Comparisons
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M A1 A2 AG1 AG2 AE1 B1 B2 B3 C1 C2 D1 D2 F1 F2 G1 G2 H2 J1 J2 Ho
p93BR029.4 - B/F
gag HMA
07/07/00
env HMA
M A2 A3 B1 B2 B3 C1 C2 C3 D1 D3 E1 E2 E3 F1 F2 G1 G2 G3 H2 Ho
02/24/200 ES7/ES8
5% PAGENO UREA
5% PAGE20% UREA
21/2 Hrs250V constant
21/2 Hrs250V constant
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Baseline specimen evaluation:Crude nucleic acid preparation (e.g., ~0.1ug cellular DNA, or
cDNA from ~0.01 ml plasma or sera)
1st Round PCR (e.g., ED3 / ED14, ~gp120)2nd Round PCR (e.g., ED5 / ED12, ~V1-V5)
Agarose gel (to quantitate product and template)
Heat, cool PCR fragments (to form heteroduplexes)5% Acrylamide gel (to assess heterogeneity)
Mix unknown with reference strains:Heat, cool PCR fragments (to form inter-sample heteroduplexes)
5% Acrylamide gel (to identify fast-migrating heteroduplexes)
Subtype determination:Patterns with references from a given subtype should be consistent
Phylogenetic clustering:Determine heteroduplex mobility relative to homoduplex
Subtype Determination with HMA
(optional)
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Heteroduplex
TrackingAssay
(Detect only heteroduplexes
formed from probe alone or with target virus population)
Probe1X
32P
Driver100X
Mix, Heat, Cool
S ingleStrands
Different Time Points or Compartments
Different PersonSame Person
Probe only
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HIV population diversifying through point mutations only
EthBr stained gel Probed with 1st time pt.
Populations sampled by DNA sequencing
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HIV population diversifying through point mutations and
length variation
EthBr stained gel
Populations sampled by DNA sequencing
2 2 codon codon deletiodeletio
nn1 1 codon codon deletiodeletio
nnno deletionno deletion
Probed with 1st time pt.
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AOGGE333JJ
BBB33JJBHB333FGBBBJHHH
F3GGGFJFFFFHHHHHHJ3BJJFGCCCEEFFC
GEEEEAAAA
ECCCCCCAAOEAAA
1%
B 4J 4.5H 53 3.5F 3G 2.5E 2C 1A 0.5O 09779859988987997100
10099
99999996781009889
OOOOOOO
homoduplexhetero-duplexes
Years Post Infection
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100,000 cell equivalents
5.4 1.8 3.9 8 4.7 3.8
V11 V13 V14 V15 V16 V18
Input TemplateCopy Number
~ Normalized copy number
16 14 27 26 28 27
V11V13 V15V16V18V14
Importance of Template Quantitation
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0
5
10
15
0 25 50 75 100 125 150
Ave
rag
e
nu
mb
er
of
un
iqu
e t
em
pla
tes
Input copy number, N
10 clones
20 clones
15 clones
5 clones
0
0.25
0.5
0.75
1P
rob
ab
ility
of
resa
mp
ling
10 clones
20 clones
15 clones
5 clones
“QUALITY” - Resampling probablilities