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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. 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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  1. HMA & HTA: Theoretical and Practical Issues

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

  3. 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)

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

  5. 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

  6. 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

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

  8. 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

  9. 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

  10. ~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

  11. Importance of Multiple Comparisons He *Please avoid “sub-subtype” designations based on affinity to different references*

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

  13. He ss He Ho Identification of a new subtype 4 RU103 vs: vs M A B C D E F 6 Ho

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

  15. Importance of the “Ho” Control 94TH135 94TH1364 94TH137 M B D E Ho B D E Ho B D E Ho He He Ho

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

  17. 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)

  18. 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)

  19. 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)

  20. 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

  21. (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

  22. 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

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

  24. 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

  25. 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

  26. “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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