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HMA & HTA: Theoretical and Practical Issues. Topics. Development of the ( env ) HMA Strategies for subtype determination Heteroduplex Tracking Assay Importance of PCR template quantitation. A. A. T. T. T. A. A. T. A. Denature,. Acrylamide. Heteroduplexes. Reanneal. Gel. T. A.

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HMA & HTA: Theoretical and Practical Issues

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Hma hta theoretical and practical issues

HMA & HTA:

Theoretical and Practical Issues


Topics

Topics

  • Development of the (env) HMA

    • Strategies for subtype determination

  • Heteroduplex Tracking Assay

  • Importance of PCR template quantitation


Hma hta theoretical and practical issues

A

A

T

T

T

A

A

T

A

Denature,

Acrylamide

Heteroduplexes

Reanneal

Gel

T

A

Homoduplexes

T

G

A

C

T

G

A

T

C

Denature,

Acrylamide

A

T

G

C

A

G

Reanneal

Gel

Heteroduplexes

Homoduplexes

C

T

G

C

A

T

A

G

T

C

Heteroduplex Mobility Assay (HMA)


Hma hta theoretical and practical issues

Urea

or

Heat

More Heat

or Urea

Melt &

Reanneal


Detection of heteroduplexes

Agarose gel

DNA (ng)

5

10

50

100

500

500

500

500

500

Cycles

35

35

35

35

35

35

35

35

25

-

-

-

-

-

-

+

-

-

Resolve

-

-

-

-

-

+

-

+

-

Heat/Cool

Acrylamide gel

Detection of heteroduplexes


Detection of mismatches without indels

Detection of mismatches (without Indels)

% mismatches

M

1.3

1.4

1.8

3.7

3.9

4.0

4.2

4.4

4.7

4.9

M


Hma hta theoretical and practical issues

Between individuals (same subtype)

Within an individual

Between individuals

(different subtype)

Spectrum of Gel Shifts (env HMA)


Hma hta theoretical and practical issues

Impact of INDELS* on

heteroduplex mobility

-

-

-

+

+

+

*INDELS - Insertions or deletions

# of bands =

(# of Distinguishable Species)2 - (# of D.S.)

3 Seq.

M

0.16

0.95

1.42

0.79

0.95

1.11

M


Hma hta theoretical and practical issues

E

D

5

E

D

1

2

E

S

7

E

S

8

E

D

3

1

E

D

3

3

V1-V2

V3 Loop

V4

V5

gp120

HIV-1 Env Gene Amplimer Sites

v

p

r

e

n

v

r

e

v

t

a

t

p

o

l

n

e

f

g

a

g

v

i

f

t

a

t

v

p

u

r

e

v

1000

2000

3000

4000

5000

6000

7000

8000

9000


Dna divergence in env regions

~Range of HMA in neutral 5% Acryl. gels

0.5kb ED31-33

1.2kb ED5-ED12

0.63kb ES7-ES8

DNA divergence in env regions

120

Within

a person or

subtype

100

Within or

between

subtypes

80

60

Between

M & O groups

40

20

0

5

10

15

20

25

30

35

40

45

50

% DNA Distance


Importance of multiple comparisons

Importance of Multiple Comparisons

He

*Please avoid “sub-subtype” designations

based on affinity to different references*


Highly divergent subtypes

He

ssDNA

Highly Divergent Subtypes

93RW003

93RW004

H

o

A

B

C

D

E

F

H

o

A

B

C

D

E

F


Identification of a new subtype

He

ss

He

Ho

Identification of a new subtype

4

RU103 vs:

vs

M

A

B

C

D

E

F

6

Ho


Efficient subtype analysis

Efficient Subtype Analysis

93TH063

93TH064

93TH062

M

B

D

E

Ho

B

D

E

Ho

B

D

E

Ho

He

He

Ho


Hma hta theoretical and practical issues

Importance of the “Ho” Control

94TH135

94TH1364

94TH137

M

B

D

E

Ho

B

D

E

Ho

B

D

E

Ho

He

He

Ho


Rapid subtyping in thailand

Rapid Subtyping in Thailand

vs. TH239B

vs. TH129E


Inter and intra subtype comparisons

WITHIN SUBTYPE COMPARISONS

A1

A1

A2

B1

B1

B2

C1

C1

C1

C2

C2

D1

D1

D2

E1

E1

E2

F1

G1

G1

G2

C3

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

A2

A3

A3

B2

B3

B3

C2

C3

C4

C3

C4

C4

D2

D3

D3

E2

E3

E3

F2

G2

G3

G3

M

Inter- and Intra-Subtype Comparisons

B2 + OTHER SUBTYPES

M

A2

B1

C2

D1

E2

F2

G2

H2

B2

1.2kb FRAGMENTS (ED5/ED12)


Hma hta theoretical and practical issues

WITHIN SUBTYPE COMPARISONS

A1

A1

A2

B1

B2

C1

C1

C1

C2

C2

D1

D1

D2

E1

E1

E2

F1

G1

G1

G2

B

1

C3

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

M

A2

B1

C2

D1

E2

F2

G2

H2

B2

B

2

M

A2

A3

A3

B3

B3

C2

C3

C4

C3

C4

C4

D2

D3

D3

E2

E3

E3

F2

G2

G3

G3

Inter- and Intra-Subtype Comparisons

B2 + OTHER SUBTYPES

0.7kb FRAGMENTS (ES7/ES8)


Hma hta theoretical and practical issues

A1

A1

A2

B1

B1

B2

C1

C1

C1

C2

C2

C3

D1

D1

D2

E1

E1

E2

F1

G1

G1

G2

WITHIN SUBTYPE COMPARISONS

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

M

A2

B1

C2

D1

E2

F2

G2

H2

B2

C4

A2

A3

A3

B2

B3

B3

C2

C3

C4

C3

C4

D2

D3

D3

E2

E3

E3

F2

G2

G3

G3

M

Inter- and Intra-Subtype Comparisons

B2 + OTHER SUBTYPES

0.5kb FRAGMENTS (ED31/ED33)


Hma hta theoretical and practical issues

p93BR029.4 - B/F

gag HMA

env HMA

M A2 A3 B1 B2 B3 C1 C2 C3 D1 D3 E1 E2 E3 F1 F2 G1 G2 G3 H2 Ho

M A1 A2 AG1 AG2 AE1 B1 B2 B3 C1 C2 D1 D2 F1 F2 G1 G2 H2 J1 J2 Ho

07/07/00

02/24/200 ES7/ES8

5% PAGE

NO UREA

5% PAGE

20% UREA

21/2 Hrs

250V constant

21/2 Hrs

250V constant


Subtype determination with hma

(optional)

Subtype Determination with HMA

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


Hma hta theoretical and practical issues

Same Person

Different Person

Probe only

Different Time Points or Compartments

HeteroduplexTrackingAssay(Detect only heteroduplexes formed from probe alone or with target virus population)

32

P

Probe

1X

Mix, Heat, Cool

S

i

n

g

l

e

S

t

r

a

n

d

s

Driver

100X


Hiv population diversifying through point mutations only

Probed with 1st time pt.

Populations sampled by DNA sequencing

HIV population diversifying through point mutations only

EthBr stained gel


Hiv population diversifying through point mutations and length variation

Probed with 1st time pt.

2 codon deletion

1 codon deletion

no deletion

HIV population diversifying through point mutations and length variation

EthBr stained gel

Populations sampled by DNA sequencing


Importance of template quantitation

V11

V13

V14

V15

V16

V18

27

16

14

27

26

28

~ Normalized copy number

Importance of Template Quantitation

V11

V13

V14

V15

V16

V18

Input Template

5.4

1.8

3.9

8

4.7

3.8

Copy Number

100,000 cell equivalents


Hma hta theoretical and practical issues

“QUALITY” - Resampling probablilities

1

20 clones

0.75

15 clones

Probability of resampling

0.5

10 clones

0.25

5 clones

0

20 clones

15

15 clones

Average number of

unique templates

10

10 clones

5

5 clones

0

0

25

50

75

100

125

150

Input copy number, N


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