HMA & HTA: Theoretical and Practical Issues

HMA & HTA:Theoretical and Practical Issues

Topics

Development of the (env) HMA Strategies for subtype determination

Heteroduplex Tracking Assay

Importance of PCR template

quantitation

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

Heat

Ureaor

More Heator Urea

Melt &Reanneal

Detection of heteroduplexes

25--

50035-+

50035+-

50035-+

5003535 3535-- ---- --

500100501035Cycles-Resolve-Heat/Cool

5DNA (ng)

Agarose gel

Acrylamide gel

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

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

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

Spectrum of Gel Shifts (env HMA)

Within an individual

Between individuals (same subtype)

Between individuals(different subtype)

% 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)

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.)

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

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

M Ho A B C D E F

Importance of Multiple Comparisons

He

*Please avoid “sub-subtype” designationsbased on affinity to different references*

He

Ho A B C D E F Ho A B C D E F

93RW003 93RW004

Highly Divergent Subtypes

ssDNA

M Ho A B C D E F

4vs6

RU103 vs:

Identification of a new subtype

He

Ho

He

ss

M HoB D E

He

He

Ho

93TH063HoB D E

93TH064HoB D E

93TH062

Efficient Subtype Analysis

M HoB D E

He

He

Ho

94TH135HoB D E

94TH1364HoB D E

94TH137

Importance of the “Ho” Control

Vs.TH239

B

Vs.TH129

E

vs. TH239B

vs. TH129E

Rapid Subtyping in Thailand

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

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

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

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

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)

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

HIV population diversifying through point mutations only

EthBr stained gel Probed with 1st time pt.

Populations sampled by DNA sequencing

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.

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

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

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