1. Theoretical and Practical Issues
HMA & HTA:
Theoretical and Practical Issues

2. Topics Development of the (env) HMA Heteroduplex Tracking Assay
Strategies for subtype determination
Heteroduplex Tracking Assay
Importance of PCR template quantitation

3. Heteroduplex Mobility Assay (HMA)
Acrylamide
Gel
Homoduplexes
Heteroduplexes
Denature,
Reanneal G C A T T C A G
Acrylamide
Gel
Heteroduplexes
Homoduplexes
Denature,
Reanneal

4. Heat
Urea or
More Heat
or Urea
Melt &
Reanneal

5. Detection of heteroduplexes
Agarose gel
Acrylamide gel 35 -
500
100 50 10
Cycles
Resolve
Heat/Cool 5
DNA (ng) 35 - +
500 35 + -
500 35 - +
500 25 -
500

6. Detection of mismatches (without Indels)
1.3
1.4
1.8
3.7
3.9
4.0
4.2
4.4
4.7
4.9 M

11. Spectrum of Gel Shifts (env HMA)
Between individuals (same subtype)
Within an individual
Between individuals
(different subtype)

13. heteroduplex mobility
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 - +

14. HIV-1 Env Gene Amplimer SitesD 5 1 2 S 7 3 8
V3 Loop V4 V5
gp120
V1-V2 v p r e n v p o l • • r e v t a t • 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

15. DNA divergence in env regions
~Range of HMA in neutral 5% Acryl. gels
120
0.5kb ED31-33
Within
a person or
subtype
1.2kb ED5-ED12
100
0.63kb ES7-ES8 80
Within or
between
subtypes 60
Between
M & O groups 40 20 5 10 15 20 25 30 35 40 45 50
% DNA Distance

16. Importance of Multiple ComparisonsHe
*Please avoid “sub-subtype” designations
based on affinity to different references*

17. Highly Divergent Subtypes
93RW003
93RW004 H o A B C D E F H o A B C D E F He
ssDNA

18. Identification of a new subtypeHe Ho ss
RU103 vs: 4 vs M A B C D E F 6 Ho

19. Efficient Subtype Analysis
93TH063
93TH064
93TH062 M B D E Ho B D E Ho B D E Ho He He Ho

20. Importance of the “Ho” ControlB D E Ho B D E Ho B D E Ho He He Ho

21. Rapid Subtyping in Thailand
vs. TH239B
vs. TH129E

22. Inter- and Intra-Subtype Comparisons
B2 + OTHER SUBTYPES M A1 A3 + A2 B1 B3 B2 C1 C3 C2 D1 D2 D3 E1 E2 E3 F1 F2 G1 G2 G3 C4
WITHIN SUBTYPE COMPARISONS M A2 B1 C2 D1 E2 F2 G2 H2 B2
1.2kb FRAGMENTS (ED5/ED12)

23. Inter- and Intra-Subtype Comparisons
B2 + OTHER SUBTYPES M A1 A3 + A2 B1 B3 B 2 1 C1 C3 C2 D1 D2 D3 E1 E2 E3 F1 F2 G1 G2 G3 B2 C4
WITHIN SUBTYPE COMPARISONS M A2 B1 C2 D1 E2 F2 G2 H2 B2
0.7kb FRAGMENTS (ES7/ES8)

24. Inter- and Intra-Subtype Comparisons
B2 + OTHER SUBTYPES M A1 A3 + A2 B1 B3 B2 C1 C3 C2 D1 D2 D3 E1 E2 E3 F1 F2 G1 G2 G3 C4
WITHIN SUBTYPE COMPARISONS M A2 B1 C2 D1 E2 F2 G2 H2 B2
0.5kb FRAGMENTS (ED31/ED33)

25. p93BR029.4 - B/F env HMA gag HMA 21/2 Hrs 250V constant 21/2 Hrs
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 B C1 C D1 D2 F1 F 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

26. 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
(optional)

27. Same Person
Different Person
Probe only 32 P
Probe 1X
Heteroduplex Tracking Assay (Detect only heteroduplexes formed from probe alone or with target virus population)
Mix, Heat, Cool S i n g l e S t r a n d s
Driver
100X
Different Time Points or Compartments

28. HIV population diversifying through point mutations only
EthBr stained gel
Probed with 1st time pt.
Populations sampled by DNA sequencing 

29. HIV population diversifying through point mutations and length variation
EthBr stained gel 
2 codon deletion
1 codon deletion
no deletion
Probed with 1st time pt.   
Populations sampled by DNA sequencing

31. Importance of Template Quantitation
V11
V13
V14
V15
V16
V18
~ Normalized copy number 16 14 27 26 28
V11
V13
V15
V16
V18
V14
Input Template
5.4
1.8
3.9 8
4.7
3.8
Copy Number
100,000 cell equivalents

33. “QUALITY” - Resampling probablilities1
20 clones
0.75
15 clones
Probability of resampling
0.5
10 clones
0.25
5 clones
20 clones 15
15 clones
Average number of
unique templates 10
10 clones 5
5 clones 25 50 75
100
125
150
Input copy number, N