LATE-PCR and Allied Technologies for Analysis of Genetic Heterogeneity in Single Cells & Single DNA Molecules

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1 Building Tools for the Analysis of Cellular Heterogeneity LATE-PCR and Allied Technologies for Analysis of Genetic Heterogeneity in Single Cells & Single DNA Molecules J. Aquiles Sanchez, Adam E. Osborne, and Lawrence J. Wangh Brandeis University October 16, 2009 NIH CEGS Grantees Meeting Huntsville, Alabama

2 The LATE-PCR Technology Platform Sample Preparation Quantitative recovery of nucleic acids from single cells PurAmp (DNA & RNA)

3 The LATE-PCR Technology Platform Sample Preparation Amplification Method Efficient amplification of single-stranded DNA PurAmp (DNA & RNA) LATE PCR

4 The LATE-PCR Technology Platform Sample Preparation Amplification Method PurAmp (DNA & RNA) LATE PCR PrimeSafe Suppresses non-specific amplification products

5 The LATE-PCR Technology Platform Sample Preparation Amplification Method Product Analysis Multiplexing PurAmp (DNA & RNA) LATE PCR Increased Detection Sensitivity PrimeSafe Direct DNA Sequencing

6 Genetic Heterogeneity at the Single Cell Level: The Hypoxia Transcriptional Response Low O 2 concentration (Hypoxia) Glycolytic enzymes Anaerobic Metabolism HIF TP53 p14 ARF HSP70 EPO VEGF Activates Hypoxia Response Programmed Cell Death Regulates Hypoxia Response Cope with hypoxic stress Erythropoieisis Angiogenesis

7 Limitations of Conventional PCR Symmetric RT-PCR Equal concentration of amplification primers detection background real-time

8 Limitations of Conventional PCR Symmetric RT-PCR Equal concentration of amplification primers Amplification stops because double-stranded DNA inhibits Taq DNA polymerase detection background real-time

9 Fluorescence Limitations of Conventional PCR Symmetric RT-PCR Equal concentration of amplification primers Co-Amplification of Abundant & Rare Targets Plateau Phase Amplification stops because double-stranded DNA inhibits Taq DNA polymerase Exponential Phase real-time detection background Cycle Number Abundant targets prevent amplification of rare targets

10 The LATE-PCR Solution Linear- After-The-Exponential (LATE) RT-PCR Unequal concentration of amplification primers end-point Limited accumulation of double-stranded DNA products does not stop PCR real-time detection background

11 Fluorescence The LATE-PCR Solution Linear- After-The-Exponential (LATE) RT-PCR Unequal concentration of amplification primers Limited accumulation of double-stranded DNA products does not stop PCR real-time detection background end-point TET-H5, FAM-Control Probe Fluorescence ( R n ) TET-H5, FAM-Control Probe Fluorescence ( R n ) Co-Amplification of Abundant & Rare Targets 1600 TET-H5 RNA HEX-N1 RNA Control DNA Target RNA I = 10 9 copies TET-H5 RNA 700 Target RNA II = 10 HEX-N1 RNA 9 copies Target RNA III = 10 Control DNA copies C 200 T = 20 = fold C T = 20 = fold 10 3 copies Cycle Number -100 Cycle Number 10 3 copies Cycle Number Hartshorn & Wangh (2009) One-step RT-LATE-PCR for mrna and Viral RNA Detection and Quantification (Methods in Molecular Biology series, The Humana Press) HEX-N1 Probe Fluorescence ( R n ) HEX-N1 Probe Fluorescence ( R n )

12 Symmetric PCR Double-stranded DNA competes with binding of detection probes

13 LATE-PCR Provides Increased Detection Sensitivity Symmetric PCR LATE-PCR Double-stranded DNA competes with binding of detection probes Single-stranded DNA products are saturated with detection probes for maximum sensitivity

14 Multiplex LATE-PCR Assays for Analysis of the Hypoxia Response in Single Cells HIF1 TP53 p14arf HIF TP53 p14 ARF HSP70 EPO VEGF HSP70 EPO VEGF

15 Optimized Multiplexed LATE-PCR Assays Using PrimeSafe HIF1 TP53 p14arf HIF TP53 p14 ARF HSP70 EPO VEGF HSP70 EPO VEGF

16 Fluorescence DNA + RNA copy number DNA+RNA copy number Quantitative Endpoint Analysis Quantitative Endpoint Analysis obtained from Ct obtained from end point 300 Cycle Number Cycle Number Quantitative Real-Time Endpoint Analysis Activation Heat of Gene shock Expression time (mins) (min)

17 Multiplex LATE-PCR Assays for Analysis of the Hypoxia Response in Single Cells HIF1 TP53 p14arf HIF TP53 p14 ARF HSP70 EPO VEGF HSP70 EPO VEGF

18 Genetic Heterogeneity at the Level of Single DNA Molecules: Mutational Load of Mitochondrial Genomes

19 Genetic Heterogeneity at the Level of Single DNA Molecules: Mutational Load of Mitochondrial Genomes Neoplasia is driven by the accumulation of genetic changes

20 Genetic Heterogeneity at the Level of Single DNA Molecules: Mutational Load of Mitochondrial Genomes Neoplasia is driven by the accumulation of genetic changes Does mitochondria undergoes random genetic changes during neoplastic progression?

21 Genetic Heterogeneity at the Level of Single DNA Molecules: Mutational Load of Mitochondrial Genomes Neoplasia is driven by the accumulation of genetic changes Does mitochondria undergoes random genetic changes during neoplastic progression? A single cell has ~ 1000 mitochondrial DNAs

22 Genetic Heterogeneity at the Level of Single DNA Molecules: Mutational Load of Mitochondrial Genomes Neoplasia is driven by the accumulation of genetic changes Does mitochondria undergoes random genetic changes during neoplastic progression? A single cell has ~ 1000 mitochondrial DNAs Detection of random, low frequency mutations requires analysis of single mitochondrial DNAs

23 A 10-plex LATE-PCR Assay for Analysis of Single Mitochondrial DNA 10 primers sets for 8 genes/regions: HV1, HV2, 12srRNA, ND1, ND4, CO1, CO2, and CytB Covering 30% of the mitochondrial genome Gene Length (bp) Amplicon Length (bp) % Gene Covered HV (495) 100 HV (408) srRNA (794) 83 16,569bp ND (584) 61 ND (434) (436) 63 CO (573) 37 CO (522) 76 Cytb (394) (378) 68

24 ABS ABS ABS ABS Amplification Products from the 10-Plex Assay 16 Cal Orange Cyb-1 12s HV1 25 Quasar ND4-1 CO Temperature Temperature 8 Fam 7 ND Cal Red 10 CO HV Cyb-2 ND Temperature Temperature

25 Sequencing of Individual Products from the 10-Plex HV1 Cyb1 HV2 Cyb2 12s ND1 CO1-1 ND4-1 CO2 ND4-2

26 Analysis of Single Mitochondrial DNAs Identifies Random Mutations Not Seen in Bulk DNA Total Cell Sample 1000 mtdna copies HV1 Gene Single Molecule Sample 1 mtdna copy HV1 Gene Osborne et al., (2009) Single-Molecule LATE-PCR Analysis of Human Mitochondrial Genomic Sequence Variations, PLoS ONE 4: e5636, 2009