III Milestone: Nanoparticle hybridization and SPR shift

5. Discussion

5.5 III Milestone: Nanoparticle hybridization and SPR shift

There has been a significant amount of research dedicated to study properties and applications of DNA functionalized with gold nanoparticles (MCKENZIE et al., 2008).

The conjugation of laser-generated gold nanoparticles with biomolecules allows their application in biotechnology research. Mirkin et al. (1996) report the first method for assembling gold nanoparticle to DNA oligonucleotides capped with thiol group (MIRKIN et al., 1996).

Nano-targeting is based on the fact that oligonucleotide probes by specific hybridization trigger gold nanoparticles on the targeted sequence, causing a nanoparticle agglomeration. Such agglomeration has a unique signal and can be distinguished by the non-hybridized probe because of an absence of agglomeration.

Triplex target sites and triplex hybridization is suitable for gold nano-targeting because of the enriched status of poly-purine sequences in eukaryotic genomes, and moreover because these sequences are interrupted by one or more base pair inversions (example in Figure 16) (XODO et al., 2001). Target sequences very close to each other result in a better AuNPs agglomeration and therefore a better signaling.

Figure 16: Ideal configuration of AuNPs-TFOs. Blue nucleotides represent triplex forming oligonucleotides made of two different specific sequences, in red a dsDNA target made by two different poly-purine sequences spaced by only one nucleotide. Gold nanoparticles are represented as red circles.

Chemical synthesis is producing nanoparticles with impurities resulting from the use of surfactants, chemical precursors and reducing agents, restricting their use in biomedicine (PETERSEN et al., 2009, BARCIKOWSKI and COMPAGNINI, 2013).

The use of laser-generated particles often leads to significantly better results in medical technology because they are usually inert, incontrast to the chemical synthesized ones (BARCIKOWSKI and COMPAGNINI, 2013, REHBOCK et al., 2013).

Labeling of AuNPs with triplex forming oligonucleotides was performed ex situ, meaning conjugation was performed after nanoparticles synthesis. This characterized the formation of conjugates formed by one AuNPs linked to multiple TFOs. An ideal model of the conjugate may be formed by a nanoparticle for each

molecule of TFO. This would maximize the number of particles agglomerated during hybridization.

TFO hybridization with condensed DNA, especially with bovine Y-chromosome specific sequence was never done before and it represents a big challenge. Gold nanoparticles influence DNA molecular kinetic, it was described that AuNPs cause a change of the dissociation enthalpy of DNA duplexes influencing hybridization (CHEN et al., 2009). A first attention was focused on AuNPs diffusion properties within condensed DNA environment, knowing that molecular diffusion in cytoplasm is only 3 to 4 times slower than in water due to molecular crowding. It was expect that diffusion in crystalline DNA structure was also less (WEISS et al., 2004, BANKS and FRADIN, 2005). Consequently, AuNPs/DNA conjugate hybridize to DNA molecules in solution with a slower kinetic. It was previously studied that ssDNA probes functionalized with AuNPs hybridize to complementary target ssDNA in a solution, but does not directly react with the DNA target, but rather it is first adsorbed onto the AuNPs surface, followed by two-dimensional diffusion until it finally hybridizes with immobilized DNA (CHEN et al., 2009).

An interesting work recently made was done using ultra small AuNPs as carriers for nucleic acids probes as therapy for cancer diseases (HUO et al., 2014). Within their study, 2nm gold nanoparticles labeled to TFO were able to transport inside the nucleus of MCF7 cells (breast cancer cell line, Michigan Cancer Foundation-7). Gold nanoparticles were used as carriers for TFO to hybridize c-myc gene and

down-70

regulating it. Huo et al, used 2 nm sized AuNPs as carriers for small DNA oligonucleotides; evaluation of such small sized particles also can be tested in sperm nano-targeting.

Gold nanoparticles were functionalized with highly reactive proteins. The protein corona (CEDERVALL et al., 2007) is typical phenomenon for which gold nanoparticles are surrounded by proteins that compete among themselves for the surface attachment. Moreover, it was described that RNA-protamine-AuNPs conjugates have a great stability because of high affinity between protamines and AuNPs (DELONG et al., 2009). Protamines affinity for nanoparticles may represent an obstacle to triplex hybridization, and this hypothesis is consistent with the low SPR shift analyzed. The agglomeration of the conjugates on the nuclei could also be due to the smaller volume of the Y-nuclei compare to the X-nuclei (VAN MUNSTER et al., 1999). Therefore, no agglomeration may be due to a specific effect of DNA but due to the reactivity of the gold nanoparticles and protamines.

The experiments described within this thesis on AuNP/TFO interaction with free DNA and sperm nuclear matrix have different results. When the conjugate is incubated with DNA, nanoparticles show a shift in SPR of 7nm for male versus female DNA. On the other hand, when the conjugate is incubated with DNA, the shift in SPR is of 1nm for male versus female DNA. The red shift deviation was not significant for either sample probes. The missing wavelength deviation denotes a significant difficulty for nanoparticles bio-conjugates to hybridize in condensed chromatin. The SPR

measurement alone is not sufficient to affirm that the AuNPs were hybridized. Due to their high reactivity with proteins (protein corona), nanoparticles can create a high background because reacting with protamines. Perhaps the shift of AuNPs/TFO after incubation with sperm nuclear matrix is due to different volume of X-and Y-chromosome bearing sperm. Since the Y-Y-chromosome bearing-sperm are smaller than X-bearing ones (VAN MUNSTER et al., 1999), nanoparticles stuck on the surface are necessarily closer to each other and this situation may influence the change of the SPR. To discharge this possibility, a double check experiment (microscopic) in order to visualize the nanoparticles in situ has to be planed. The conjugated AuNPs / TFO can be labeled to fluorochromes to be used in triplex in situ hybridization. Such experiment must be taken in consideration for future studies.

A second interpretation of the failure of SPR deviation between the sex samples is that AuNPs are too small to give a significant red shift signal. Therefore, bigger nanoparticles have to be tested.

A sine qua non condition for the future of sperm nano-targeting is that AuNPs bio-conjugates pass through the sperm membranes. AuNPs internalization of the conjugate in the sperm is not argued in this thesis. Literature shows that there is no penetration of AuNPs alone or conjugated to cell penetrating peptides and/or to DNA probes (TAYLOR et al., 2014, TIEDEMANN et al., 2014). However, Barchanski et al.

(2015) demonstrated that bio-conjugated AuNPs do penetrate into spermatozoa but depending on its plasma membrane status (BARCHANSKI ET AL, 2015).

72 5.10 Future outlook

Future research has to focus on different topics. First, since triplex FISH ranking was improved by using a synergy of different probes, a step forward can be made by using more probes (about 20), all conjugated with fluorochromes. It is also important to program a method to obtain best results in the red shift. The variables that act are different:

• Gold nanoparticle size. The synthesis of smaller nanoparticles can affect favorably hybridization triplex as shown by Huo et al (HUO et al., 2014), on the contrary it may represent a disadvantage for an efficient SPR shift. Larger particles also would present pros and cons: on the one hand they may increase the shift SPR but inhibit triplex hybridization and may also present problems in the penetration of sperm nuclei at the same time.

• Perform a gold nanoparticle triplex FISH, using AuNPs labeled to fluorescent probes.

• Ratio probe/nanoparticles. The synthetic method chosen for the production of conjugate was the ex situ conjugation, meaning gold nanoparticles are conjugated to TFO probes after their synthesis. This has characterized the formation of conjugates formed by a single nanoparticle linked to multiple TFOs. An ideal model of the conjugates may be formed by one single nanoparticle for each molecule of TFO. This would maximize the number of particles agglomeration during hybridization.

• Number of probes. The approach of the present this was to screen probes and choose the best one. Increasing number of probes and repeatedly using these

in greater amounts on bovine Y-chromosome bearing sperm, could lead to higher agglomeration and to a greater shift in SPR.

• Perform a triplex FISH with probes linked both to nanoparticles and to fluorochromes, in order to identify and verify proper hybridization of the conjugate.

More research is needed to understand the reason why bovine Y-chromosome bearing sperm are rich of poly-purine sequences and to compare these sequences to all autosomes and check possible functional relations. It would be also interesting to compare H-DNA in male and female subjects to understand if intra-molecular triplexes are involved for development of male phenotype.

74 6. Summary

Roberto Mancini

Detection of Y-chromosome bearing bovine sperm using laser-generated gold nanoparticle bio-conjugates

This study describes two innovative aspects of molecular biology, triplex-forming-oligonucleotides (TFOs) alone and labeled to gold nanoparticles. The thesis aims to develop a novel approach for sperm gene targeting and its applicability to sperm sex sorting, based on vital genetic sequences detection.

Besides the use of other markers that recognize DNA, triplex-forming oligonucleotides represent an alternative approach for specific gene detection suitable for vital cells. The success of the triplex-based chromosome targeting is governed by the specific oligonucleotide binding.

Within this thesis an abundance of triplex target sites were found on the bovine chromosome, which makes the triplex hybridization a valid alternative for Y-chromosome bearing sperm detection.

Three candidate TFOs, with a length of circa 20 bases, were analyzed for hybridization efficiency on the bovine Y-chromosome. Better triplex hybridization efficiency was achieved using more probes simultaneously.

Not all Y-chromosome sperm nuclei were detected by TFOs, most likely due to the intrinsic condensed DNA structure.

This study confirms that TFOs with a low content of cytosine have a higher hybridization temperature.

To define TFOs hybridization for future studies, it is necessary to consider further variables, such as time of incubation of target DNA and TFO probes, temperature, TFO’s sequence and 5- methylcytosine content, buffer and bovine individual sequence variations.

Gold nanoparticle conjugated with TFOs can hybridize to genomic DNA free in solution and lead to a surface plasmon resonance shift. The use of the conjugate in sperm chromatin resulted in a non-significant signaling.

More studies are needed to identify different sized AuNPs (from 2 nm to 50 nm) conjugates and their potential use in a condensed chromatin structure.

76 7. Zusammenfassung

Detektion Y-Chromosomen boviner Spermien durch laser-generierte Gold Nanopartikel-Biokonjugate

Diese Studie beschreibt zwei innovative Aspekte der molekularen Biologie, Triplex-bildende Oligonukleotide (TFOs) allein und konjugiert mit Gold-Nanopartikeln. Ziel der Arbeit war es, eine neue Methode für die spezifische Genommodifikation von Spermien und deren Anwendbarkeit beim Sperma-Sexing zu entwickeln, die auf der Lebend-Detektion von Gensequenzen basiert.

Neben der Nutzung anderer DNA-detektierender Marker stellen Triplex-bildende Oligonukleotide eine alternative Methode für die spezifische Gendetektion in vitalen Zellen dar. Das Gelingen der Triplex-basierten Chromosomen-Markierung wird durch die Bindung spezifischer Oligonukleotide erzielt. In dieser Arbeit wurden mehrere Zielsequenzen für Triplex-Sonden auf dem bovinen Y-Chromosomen gefunden.

Damit ist die Triplex-Hybridisierung auch für die Detektion von Y-Chromosomen-tragende Spermien eine zuverlässige Methode .

Drei ausgewählte TFOs, die eine Länge von circa 20 Basen aufwiesen, wurden in Hinblick auf ihre Hybridisierungseffizienz mit bovinen Y-Chromosomen analysiert.

Eine bessere Hybridisierungsfähigkeit der Triplex-Sonden wurde durch die simultane Nutzung mehrerer Sonden erreicht.

Nicht alle Y-Chromosome tragenden Spermien konnten mit TFOs markiert werden.

Dieses ist höchstwahrscheinlich auf den DNA-Kondensierungsrad zurückzuführen.

Diese Studie bestätigt, dass für TFOs mit niedrigem Cytosingehalt eine höhere Hybridisierungstemperatur notwendig ist.

Um das Hybridisierungspotential von TFOs für zukünftige Studien festzulegen, ist es nötig, weitere Variablen zu berücksichtigen, beispielsweise die Koinkubationszeit für die Ziel-DNA mit den TFO-Sonden, die Inkubationstemperatur, die Sequenz der TFOs und ihr Methylcytosingehalt, die Art der Puffer und individuelle Sequenzvariationen zwischen verschiedenen Bullen.

Gold-Nanopartikel, die mit TFOs konjugiert sind, können mit genomischer DNA, die frei in Lösung ist, hybridisieren und führen zu einer Plasmon-Resonanz-Verschiebung (SPR). Die Anwendung dieser Konjugate mit Spermienchromatin führte nicht zu einem signifikanten Signalunterschied. Weitere Studien sind nötig, um Konjugate mit Gold Nanopartikeln verschiedener Größe (2 bis 50 nm) und ihre mögliche Anwendbarkeit für kondensierte Chromatinstrukturen zu identifizieren.

78 8. Appendix

7.1 Abbreviations

AuNPs Gold nanoparticles

CRISPR Clustered regularly interspaced short palindromic repeats CRM Chemical reduction method

CSU Central sorting unit

SsDNA Single stranded deoxyribonucleic acid DsDNA Double stranded deoxyribonucleic acid H-DNA H structure deoxyribonucleic acid B-DNA B structure deoxyribonucleic acid EMSA Electrophoretic mobility shift assay EGFP Enhanced green fluorescent protein LNA Locked nucleic acids

MCF-7 Michigan cancer foundation - 7 MSY Male specific region

MACS Magnetic activated cell sorting MEMS Micro electro-mechanical system NMR Nuclear magnetic resonance

PGD Pre-implantation genetic diagnostic PMT Photo multiplier tube

RT Room temperature

TALEN Transcription activator–like effector nuclease TCEP Tris(2-carboxyethyl) phosphine

TFO Triplex forming oligonucleotide TTS Triplex target sites

SPR Surface plasmon resonance SSC Saline-sodium citrate

UNA Unlocked nucleic acids

79 7.2 List of tables

Table 5: TTS in bovine Y-chromosome. List of triple target sequences and relative number of repeats all along the Y-chromosome.

1. AAAGGAGGAGAAGGAAAAGGAGGGG; 142 2. GGAAAGGGAGGAGAGAGGAGAGGGA; 2 3. AGGGGAAGGAGAGAGAAGAGGGGGA; 70 4. GAGGGGGAGGGGAGAGGGGAAGGAA; 2 5. GGGGAAGGAGAGAGAAGAGGGGGAA; 70 6. GGGGGAGAGGGAGAGAAGAAGGGAA; 2 7. AAGGGGAAGGAGAGAGAAGAGGGGG; 75 8. AGAGGGAGGGAGGGAGGGAGGGAGG; 67 9. AAAGGGGAAGGAGAGAGAAGAGGGG; 75 10. AAAGGGGAGGGGGGAGAAAGAAAGA; 1 11. AAGGGGAAGGAGAGAGAAGAGGGGA; 75 12. AGAAAGGGAGGGGGAAGGGAGAGGG; 1 13. GGGGAAGGAGAGAGAAGAGGGGGAG; 70 14. AGAAGGGAGAAGAGGGGAGAAAGGG; 1 15. AGGGGAAGGAGAGAGAAGAGGGGAG; 70 16. AGGGAGAGAAGAAGGGAAAGGGAGA; 1 17. AAAGGGGAAGGAGAGAGAAGAGGAG; 74 18. AGGGGAAGAAGGAAGGGGAGAAGGG; 1 19. AAGGGGAAGGAGAGAGAAGAGGAGG; 74 20. GAAAAGGGAGGGGAGAAGAGAGGGA; 1 21. GGGGAAGGAGAGAGAAGAGGGGAGG; 70 22. GAAGGGAGAAGAGGGGAGAAAGGGG; 1 23. AGGGGAAGGAGAGAGAAGAGGAGGA; 69 24. GAGGGGAAGGGGAGAAAGGAGAGGA; 1 25. GGAGGGGAGGGGAGGAAGGAGGGAG; 122 26. GGAGAGAAAGAAGGGGAGGGGAGGG; 1 27. GGGGAAGGAGAGAGAAGAGGAGGAG; 69 28. GGAGGGGAAAAGAGGGAGGAGGAGA; 1 29. AAGGGAGGAGGAGGGGGAGGGGGAG; 72 30. GGAGGGGGAAGGGAGAGGGGGGGAA; 1 31. AGGGAGGAGGAGGGGGAGGGGGAGG; 72 32. GGGAAAGGGGAGAAGGGGAAGGGAG; 1 33. GGAGGAGGAGGGGGAGGGGGAGGGG; 76 34. GGGGAAGAAGGAAGGGGAGAAGGGG; 1 35. GAGGAGGAGGGGGAGGGGGAGGGGG; 76 36. GGGGGGAGAAGGGGAGGGGGGAGAA; 1 37. GGGAGGAGGAGGGGGAGGGGGAGGG; 76 38. GGAGGGAGGGAGGGAGGGAGGGAGG; 67 39. GGGAGGGGAGGAAGGAGGGAGGAGG; 133 40. GAAGAGAGGGAAGGGAGGGGAGGGG; 2 41. GGAGGGGAGGAAGGAGGGAGGAGGG; 133 42. GAGGGGAGGAAGGGAGGGGAGAAGG; 2 43. GAGGGGAGGGGAGGAAGGAGGGAGG; 130 44. GAGGGGGAGGGGGAGGGGGAGGGGG; 8

45. AGGAGGAGGGGGAGGGGGAGGGGGA; 76 46. GGGAGGGAGGGAGGGAGGGAGGGAG; 67 47. GGGGAGGGGAGGAAGGAGGGAGGAG; 133 48. AAAAGGGAGGGGAGAAGAGAGGGAA; 1 49. AGGGGAGGGGAGGAAGGAGGGAGGA; 130 50. AGAAGGGGGAGGGGAGAAAGGGGAG; 1 51. GGGGAGGAAGGAGGGAGGAGGGAGG; 134 52. AGGGAAAGGGGAGAAGGGGAAGGGA; 1 53. AGGGAAAAAGGGAGAAGGAGGGGGA; 5 54. AGGGGGAGAAAGAGAAGGGAGAGGA; 1 55. AAGGGGGAGGGGGGAGAAGGGGGAG; 4 56. GAGAAGGGGAAGGGGGAAGGGAGAA; 1 57. GAAGGGGGAGGGGGGAGAAGGGGGA; 5 58. GAGGGGAGAAAGGGGAGGGGAAGGA; 1 59. AGGGGGAGGGGGGGGAAAGGGAGGA; 1 60. GGAAGAAGGGGGAGGGGGAGAAAGA; 1 61. GAGGGGGAGGGGGGGGAAAGGGAGG; 1 62. GGGGGAGAAAGAGAAGGGAGAGGAA; 1 63. GAGAGAGGGAAGAGGGGGGGAAAAG; 2 64. GGAGAGAAGGGGGAGGGGGAGGGAA; 2 65. AGGGGGAGGGGGGAGAAGGGGGAGG; 4 66. GGAGGGGGAGGGGGAGGGGAGAGGG; 7 67. GGGGAGGGGGGAGAAGGGGGAGGGG; 6 68. GAGGGAGGGAGGGAGGGAGGGAGGG; 67 69. GGGGGAGGGGGGAGAAGGGGGAGGG; 4 70. GAGGGGGAGGGGGAGGGGGAGGGGA; 8 71. GGGAGGGGGGAGAAGGGGGAGGGGG; 6 72. AGGAGGGGGAGGGGGAGGGGGAGGG; 8 73. AGAAGGGGGAGGGGGGAGAAGGGGG; 5 74. GGGGAGGGGGAGGGGAGAGGGGAAG; 3 75. GGGAGGGGAGAAGGGGGAGGGGGAG; 9 76. AGGAGAGAAGGGGGAGGGGGAGGGA; 2 77. GGGGGAGGGGGGGGAAAGGGAGGAA; 1 78. GAGAAGAAAAGGGGGAGGGAGGAGA; 2 79. GGAGGGGGGAGAAGGGGGAGGGGGG; 6 80. GAGAGAGAGAGAAGGGGAGGGGAGA; 3 81. AAGGGGAGGGGAGAAGGGGGAGGGG; 9 82. GGAGGGGGAGGGGGAGGGGGAGGGG; 8 83. AGAAGGGGGAGGGGGGAGAAGGGGA; 5 84. GGGAGGGGGAGGGGGAGGGGGAGGG; 8 85. GAGAAGGGGGAGGGGGGAGAAGGGG; 5 86. GAGGGGGAGAAAGAGAAGGGAGAGG; 1 87. AGGGGAGGGGGGAGAAGGGGGAGGG; 4 88. GAGGGGGGAGAAGGGGAGGGGGGAG; 1 89. GAAGGGGGGGGGGGGGGAAAAAAAA; 15 90. GGAGGGGGGAGAAGGGGAGGGGGGA; 1 91. AGGGGAGGGGAGAAGGGGGAGGGGG; 9 92. GGGAGGGGAGAAGAGAGGGAAGGGA; 1 93. GGGGAGGGGAGAAGGGGGAGGGGGA; 8 94. GGGAGGGGAGGGAAGAAGGGGGAGG; 1

95. AGGAGGGGAAGGGGAGAAGAGGGGA; 5 96. GGGGAGAAGGGGAGGGGGGAGAAGG; 1 97. GAAGGGGGAGGGGGGAGAAGGGGAG; 5 98. GGGGAGAGGGGGAGGGGAAAAGGGG; 1

99. GGAGAAGGGGGAGGGGGGAGAAGGG; 5 100. GGGGAGGGGAGGGAAGAAGGGGGAG; 1 101. GGGAGGAAGGAGGGAGGAGGGAGGA;

168

102. GGGAGGGGGAGGGGGAGGGGAGAGG; 7

103. GAGGGGAGGGGAAGAAGGGAGAGGA; 7 104. AAAAGGGGAGAGGAAGGGGAGAAGG; 1 105. AAGGGGGAGGGGGGAGAAGGGGAGG; 4 106. GAAAGGGGAGAAGGGGAAGGGAGAG; 1 107. GGGAAGGGGAGAAGAGGGAAGGGGA; 4 108. GAGAAGAGAGGGAGAAGGGGAGAGG; 1 109. GGAGGGGAGGGGAAGAAGGGAGAGG; 7 110. GAGAAGGGGGAGGGGAGAAAGGGGA; 1 111. GGGGAGAGGGGAGAAGGGGGAGGGG; 6 112. GAGAGAAAGAAGGGGAGGGGAGGGA; 1 113. GGAGGGGAAGAAGGGAGAGGAGGGA; 4 114. GAGGGAAAGGGGAGAAGGGGAAGGG; 1 115. AGGGGGGAGAAGGGGGAGGGGGGAG; 6 116. GAGGGGAAAAGAGGGAGGAGGAGAA; 1 117. GAGGGGGGAGAAGGGGGAGGGGGGA; 6 118. GGAGGGGGAGAAAGAGAAGGGAGAG; 1 119. GGGAGAGGGGAGAAGGGGGAGGGGA; 6 120. GGGAAGAAGGGGAGGGGGAGAAGGA; 1 121. AAGAGAGAGGGAAGAGGGGGGGAAA; 2 122. GGGAGGGGGAAGGGAGAGGGGGGGA; 1 123. AGGGGAAGGGAAAAGGGAGGGGAGA; 2 124. GGGGAAGAAGGGGAGGGGGAGAAGG; 1 125. GGGAAGGGAAAAGGGAGGGGAGAAG; 2 126. AAGAGAGGGAAGGGAGGGGAGGGGA; 2 127. GGGGAAGGGAAAAGGGAGGGGAGAA; 2 128. AGAAGGGGAGGGGGAGGGGAGAAGG; 3 129. AAGGGGAGGGGGGAGAAGGGGGAGG; 4 130. GGGGAGGGGGAGGGGGAGGGGAGAG; 7 131. GGAGAGGGGAGAAGGGGGAGGGGAG; 6 132. AAAGGGGAGAAGGGGAAGGGAGAGA; 1 133. GGGGAGAAGGGGGAGGGGGGAGAAG; 6 134. AAGAAGGGGGAGGGGGAGAAAGAGG; 1 135. AGGGAGGAAGGGAGAAGGAGGGGGA; 7 136. AGAGGGGAGAAAGGGGAGGGGAAGG; 1 137. GGGAGGGGAGGGGAAGAAGGGAGAG; 7 138. AGGGAGGGGGAAGGGAGAGGGGGGG; 1 139. GGGGGAGAAGGGGGAGGGGGGAGAA; 6 140. AGGGGGAAGGAGAAGGGAGAAGAGG; 1 141. GGGGGAGAAGGGGGAGGGGGGAGAG; 6 142. GAGAGAGAAGGAAAGGGGAGAGAGA; 1 143. AGGGGGAAGGGGAGAAGGGGGAGGG; 4 144. GGAAGAAGGGGAGGGGGAGAAGGAG; 1

145. GAAGGGGAGGGGGGAGAAGGGGGAG; 4 146. GGGAGGGGGAGAAAGGGAGAGGGAA; 1 147. GGAAGGAGAGAGAAGAGGGGAGGGA; 8 148. GGGGGAGAAGGGGAGGGGGGAGAAG; 1 149. AGAGGGGAGAAGGGGGAGGGGAGAA; 6 150. GGGAGGAGGAGAAGGGGGAGGGGGG; 4 151. AGGGGAGAAGGGGGAGGGGAGAAAG; 6 152. GGGGGAGGGGGAGGGGGAGGGGAGA; 7 153. GGGGGGAGAAGGGGGAGGGGGGAGA; 6 154. AGGGGAGGGGAGGAGGAGGGGAAGG; 2 155. GGGAAGGAGAGAGAAGAGGGGAGGG; 8 156. AAGAAGGGGAGGGGGAGAAGGAGGG; 1 157. GGGAGAAGGGGGAGGGGGGAGAAGG; 5 158. AGAAGGGGGAGGGGGAGAAAGAGGG; 1 159. AGGGAGGGGAGGGGAAGAAGGGAGA; 7 160. AGGGAGAGAAGAAGAGAGGGAAGGG; 1 161. AAGGGAAAAGGGAGGGGAGAAGAGA; 2 162. GAGAGAGAAGGGGAGGGGAGAAGGG; 1 163. AGAGAGAGGGAAGAGGGGGGGAAAA; 2 164. GGAGAAGAGAGGGAGAAGGGGAGAG; 1 165. AGAAGGGGAAGGGGAGAAGAGGGGA; 4 166. GGAGGGGAAGAAGGGGAGGGGGAGA; 1 167. AGAAGGGGAGGGGGGAGAAGGGGGA; 4 168. GGGAGAAGGAAGGAGGGGAAGAAGG; 1 169. AGGAGAAGGGGGAGGGGGGAGAAGG; 4 170. GGAGGGGGAGGGGAGAGGGGAAGGA; 2 171. AGGGGAAGAAGGGAGAGGAGGGAAA; 4 172. AAGGGAGGGGGAAGGGAGAGGGGGG; 1 173. AGGGGGAGGGGGGAGAAGGGGAGGG; 4 174. AGAGAAAGAAGGGGAGGGGAGGGAA; 1 175. GAGGGGAGAAGGGGGAGGGGAGAAA; 6 176. AGAGAAAGGAAGGGGGAGGGAGGAG; 1 177. GAGAGGGGAGAAGGGGGAGGGGAGA; 6 178. AGGGGAAAAGAGGGAGGAGGAGAAG; 1 179. GAGGGAGGAAGGGAGAAGGAGGGGG; 6 180. AGGGGAAGAAGGGGAGGGGGAGAAG; 1 181. GAGGGGAAGAAGGGAGAGGAGGGAA; 4 182. GAAAGGGAGGGGGAAGGGAGAGGGG; 1 183. GGAGGGGAAGGGGAGAAGAGGGGAG; 4 184. GAAGAAGGGGAGGGGGAGAAGGAGG; 1 185. GGGGGAAGGGGAGAAGGGGGAGGGA; 4 186. GAAGAAGGGGGAGGGGGAGAAAGAG; 1 187. GAAGGGGAGGGGAGAAGGGGGAGGG; 6 188. GAAGGAGAAGGGAGAAGAGGGGAGA; 1 189. GAAGGGAAAAGGGAGGGGAGAAGAG; 2 190. GGAGAAGGGAGAAGAGGGGAGAAAG; 1 191. GGAAGGGAAAAGGGAGGGGAGAAGA; 2 192. GGGAGAGAAGAAGAGAGGGAAGGGA; 1 193. GGGAGAGGGAAGGGAGAAGGGGGGA; 3 194. GGGAGGGGGAGAAAGAGAAGGGAGA; 1

195. GGGGGAGGGGGGAGAAGGGGAGGGG; 4 196. GGGGAGAAGGGGGAGAGGGGGAGGG; 1 197. AGGGGAGGGGAAGAAGGGAGAGGAG; 5 198. AGGGAGGGAGGAAGGGAGAAGGAGG; 4 199. GGGAGGGGAAGAAGGGAGAGGAGAG; 5 200. GGAGGAGGGGGAGGGGGAGGGGGAG; 6 201. GAAGGAGAGAGAAGAGGGGAGGGAG; 7 202. GAGAAAAGGGAGGAGGGAGAAAGAG; 2 203. GGGGAGGGGAAGAAGGGAGAGGAGA; 5 204. AAGGGAGAGAAGAAGAGAGGGAAGG; 1 205. AGGAAGGAGGGGAAGAAGGGAGAGG; 4 206. AGGGGAGGGGAGGGAAGAAGGGGGA; 1 207. GAAGGGGAAGGGGAGAAGAGGGGAG; 4 208. GAGAAAGAAGGGGAGGGGAGGGAAG; 1 209. GAGGAGAAGGGGGAGGGGGGAGAAG; 4 210. GAGAGAGGAAGAAAAGAGGGAGAGG; 1 211. GGAAGGGAGGGGAGAAGGGGGAGAG; 4 212. GGAGGGGGAGAAAGGGAGAGGGAAG; 1 213. GGGAGGGGGGAGAAGGGGAGGGGGG; 4 214. GGGGAGAAGGAAGGAGGGGAAGAAG; 1 215. GGGGAGGGGGGAGAAGGGGAGGGGG; 4 216. GAGGGGAGGGGAGGAGGAGGGGAAG; 2 217. AAGGAGAGAGAAGAGGGGAGGGAGG; 7 218. GAGGGAGAGGGAGAGGGAGAGGGAG;

219. AGGAGGGGAAGAAGGGAGAGGAGGG; 4 220. GAGGAGGGGGAGGGGGAGGGGGAGG; 6 221. GAAGGAGGGGAAGAAGGGAGAGGAG; 4 222. AAAGGGAGGGGGAAGGGAGAGGGGG; 1 223. GAGAAGGGGAGGGGGGAGAAGGGGG; 4 224. AGAGAGAGAAGGAAAGGGGAGAGAG; 1 225. GGGGAGAAGGGGGAGGGGAGAAAGG; 5 226. GAGAGAGAAGGAGAGGAGAGAAGGG; 1 227. GGAAGGGGGAGGAAGGGGAAGGAAA; 3 228. GGAAGAAGGGAGAGGAGAGGGAGAG; 1 229. AAGGAAGGAGGGGAAGAAGGGAGAG; 4 230. GGGGGAGAAAGGGAGAGGGAAGGGA; 1 231. AAGGAGGGGAAGAAGGGAGAGGAGG; 4 232. GAGAGAAGGGGGAGGGGGAGGGAAG; 2 233. GGAAGGAGGGGAAGAAGGGAGAGGA; 4 234. AGGGGGAGGGGGAGGGGGAGGGGGA; 6 235. GGGGAGGGGGGGGAAAGGGAGGAAG; 1 236. AAGGAGAAGGGAGAAGAGGGGAGAA; 1 237. AGAGAGGGAAGAGGGGGGAGGGGAA; 2 238. AAGGGGAGAAGGGGAAGGGAGAGAA; 1 239. AGGAAGGGGGAGGAAGGGGAAGGAA; 3 240. AGAGAGAGAAGGAGAGGAGAGAAGG; 1 241. GAGGAAGAGAGAAGGGGAAGAAAGG; 3 242. AGGAGAAGGGAGAAGAGGGGAGAAA; 1

243. GGGAAGGGGAGAAGGGGGAGGGAAG; 3 244. AGGGGAGGGGGAGAAGGGAGGGGAG; 1 245. AAGGGGAAGGGGAGAAGAGGGGAGG; 4 246. GAGGGGAAGAAGGGGAGGGGGAGAA; 1 247. AGGAAGGGAGGGGAGAAGGGGGAGA; 4 248. GGAGAGGAGAGAAGGGGGAGGGGGA; 1 249. AGGGGAAGAAGGGAGAGGAGAGGGA; 4 250. GGGGAGAAAGGGAGAGGGAAGGGAG; 1 251. AGGGGAAGGGGAGAAGAGGGGAGGG; 4 252. GGGGAGAAAGGGGAGGGGAAGGAGG; 1 253. GGAGAAGGGGAGGGGGGAGAAGGGG; 4 254. GGGGGAGAGGGGGAGGGGAAAAGGG; 1 255. GGAGGAGAAGGGGGAGGGGGGAGAA; 4 256. AGAGAGAGAGAGAAGGGGAGGGGAG; 3 257. GGAGGGGAAGAAGGGAGAGGAGAGG; 4 258. GGGGGAGGGGGAGGGGGAGGGGGAG; 6 259. AGAAGGGGAGGGGAGAAGGGGGAGG; 5 260. AAAGGGGAGGGGGGGAGGGGGAAAG; 1 261. AGGAAGGAAGGGAGAAGGAGGGGGA; 5 262. AGAGAAGGAGAGGAGAGAAGGGGGA; 1 263. GAGGGGAAGAAGGGAGAGGAGAGGG; 4 264. AGAGAGAAGGAAAGGGGAGAGAGAG; 1 265. GGAAAGAAGGGAGGGGAGGGGAAGA; 4 266. GAAGGAGAGGAGAGAAGGGGGAGGG; 1 267. GGGAGAAGGGGAGGGGGGAGAAGGG; 4 268. GAGAAGGAGAGGAGAGAAGGGGGAG; 1 269. AAGGGAGGGGAGAAGGGGGAGAGGG; 3 270. GAGGGAGAGAAGAAGGGAAAGGGAG; 1 271. AGGGAGGGGAGAAGGGGGAGAGGGG; 3 272. GGAAGAGAGAGGAGAAGAGAGGGAG; 1 273. GAAGGGGAGAAGGGGGAGGGAAGGA; 3 274. GGAGAAGGGGGAGGGGAGAAAGGGG; 1 275. GGAAGGGGAGAAGGGGGAGGGAAGG; 3 276. GGGAAGGAGAAGGGAGAAGAGGGGA; 1 277. GGGGAAGGGGAGAAGGGGGAGGGAA; 3 278. GGGGAAGGAGAAGGGAGAAGAGGGG; 1 279. GAAGGAAGGAGGGGAAGAAGGGAGA; 4 280. AGAGAGAGAGAAGGGGAGGGGAGAA; 2 281. GGGGAAGGGGAGAAGAGGGGAGGGG; 4 282. GGGAGGGGGAGGGGAGAGGGGAAGG; 2 283. GGGAGGAAGGGGAAGGAAAAGAAGG; 3 284. GGGGAGGGGGAGGGGGAGGGGGAGG; 6 285. GGGAGGGGAGAAGGGGGAGAGGGGG; 3 286. AGGGGGAGGGGGAGGGGGAGGGGAG; 6 287. GGGGGAGGAAGGGGAAGGAAAAGAA; 3 288. AGAAGGAGAGGAGAGAAGGGGGAGG; 1 289. GGGGAAAGAAAGGGAGGAAGAAAGG; 5 290. AGGAGAGGAGAGAAGGGGGAGGGGG; 1 291. AGGGAAAAGGGAGGGGAGAAGAGAG; 2 292. GGAAGGAGAAGGGAGAAGAGGGGAG; 1

293. AGGGGAGAAGAGAGGGAAGGGAGGG; 2 294. GGGAGAAGGGGGAGGGGAGAAAGGG; 1 295. GGGAAGGGAGAAGAGGGAAAGGGAG; 2 296. GGGGAAAAGAGGGAGGAGGAGAAGG; 1 297. GGGGAGAAGAGAGGGAAGGGAGGGG; 2 298. GGGGGAAGGAGAAGGGAGAAGAGGG; 1 299. AGGGAGAGGGAAGGGAGAAGGGGGG; 3 300. AGAGAGAGAGAGAGAGAGGGAGGGA;

609

301. GGAGGGGAGAAGGGGGAGAGGGGGA; 3 302. AGAGAGAGAGAGAGAGAGGGAGAGG;

609

303. GGGGAGGAAGGGGAAGGAAAAGAAG; 3 304. GAGAGAGAGGGAGAGGGAGAGGGAG;

305. GAGGAAGGGAGGGGAGAAGGGGGAG; 4 306. AGGGAGGGAGAGAGAGAGAGAGAGA;

610

307. GGAGGGAGGAAGGGAGAAGGAGGGG; 6 308. GGAGAGGGAGAGAGAGAGAGAGAGA;

612

309. GAAGGGAGGGGAGAAGGGGGAGAGG; 3 310. AGAAAGAAGGGGAGGGGAGGGAAGA; 1 311. GGAAGGGGGAGGAAGGGGGAGGAAG; 3 312. AGGAAGAGAGAGGAGAAGAGAGGGA; 1 313. AGAGGGAGAGAAGAAGGGGAAGGGA; 2 314. AGGGGGAGAAAGGGAGAGGGAAGGG; 1 315. AGGGGAGGGGAAAAGGGAGAGAAGA; 2 316. GAAGAGAGAGGAGAAGAGAGGGAGA; 1 317. GAGGGGAGGGGAAAAGGGAGAGAAG; 2 318. GAGGGGGAGAAAGGGAGAGGGAAGG; 1 319. GAGGGGGAAGGGGAGAAGGGGGAGG; 2 320. GGAGGGAGGAAGAGAGAAGAAGGGG; 1 321. GGGAGGGGAGAAGAGGGAAAGGGAG; 2 322. GGGGGAGGGGGAGGGGAGAGGGGAA; 3 323. GAAAGAAGGGAGGGGAGGGGAAGAA; 4 324. AGAGAGAGAGAGAGAGAGAGGGAGG;

741

325. AAGGGGAGAAGGGGGAGGGAAGGAA; 3 326. AGAGAGAGAGAGAGAGAGAGGGAGA;

741

327. AGGAAGGGGGAGGAAGGGGGAGGAA; 3 328. AGAGAGAGAGGGAGAGGGAGAGGGA;

329. GAAGGGAGGGGAGGGGAAGAAGGGA; 4 330. AGAGGGAGAGAGAGAGAGAGAGAGA;

743

331. AGAAGGGAAAGGAGGGGGAGAAGGG; 1 332. GGAGGGAGAGAGAGAGAGAGAGAGA;

742

333. GAGGGGAAGGGAAAAGGGAGGGGAG; 1 334. AAGGAGAGGAGAGAAGGGGGAGGGG; 1 335. GAAGGGGGAAGGGAGAAGAGGGAAA; 2 336. AGAAGGAGGAAGGAGAGAGAAGGGG; 1 337. GGGGAAGGGAGAAGAGGGAAAGGGA; 2 338. GAGAGAAGGAGAGGAGAGAAGGGGG; 1 339. GGGGGAAGGGAGAAGAGGGAAAGGG; 2 340. GGGAGAGAGAGAAGGGAAGGAGAGA; 1 341. AAGGAGGAGGAGGAAAGGGAAGGGG; 5 342. AGAGAGAGAGAGAGAGAGAGAGGGA;

870

343. AAGGGAGGGGAGGGGAAGAAGGGAG; 4 344. GAGGGAGGGAGGAAGGGAGAAGGAG; 4 345. AGAAGGAAGGAGGGGAAGAAGGGAG; 3 346. GAGAGAGAGAGGGAGAGGGAGAGGG;

347. AGAGGAGGGGGAGAAAGGAGAGGAA; 2 348. AGGGAGAGAGAGAGAGAGAGAGAGA;

874

349. AGGGAGGAAGGGGGAGGAAGGGGGA; 2 350. GAGAGAGAGAGAGAGAGAGGGAGGG;

665

351. GAAGAGGGGGGAGGGGAAGAAGGGA; 2 352. AGAGAGAGAGAGAGAGAGAGAGAGG;

1002

353. GGAGGGGAGAAGAGAGGGAAGGGAG; 2 354. GAGAGAGAGAGAGAGAGAGGGAGAG;

665

355. GGGAAGAAGGGAGAGGAGGGAAAAG; 2 356. GGAGAGAGAGAGAGAGAGAGAGAGA;

1006

357. GGGAGGAAGGGGGAGGAAGGGGGAG; 2 358. AAGAAGGAGGAAGGAGAGAGAAGGG; 1 359. AAAGAAGGGAGGGGAGGGGAAGAAG; 4 360. AGAGAGAAGGAGAGGAGAGAAGGGG; 1 361. AAGGGAGAGGGAAGGGAGAAGGGGG; 3 362. AGGGGGAGAGGGGGAGGGGAAAAGG; 1 363. AAGAAGGGAGGGGAGGGGAAGAAGG; 4 364. GAGAGAGAGAAGGAAAGGGGAGAGA; 1 365. GAGAAGGGGAGGGGAGAAGGGGGAG; 4 366. GGGGAGAGAGAGAAGGGAAGGAGAG; 1 367. GAGGAAGGAAGGGAGAAGGAGGGGG; 4 368. GGGAGGGAGAGAGAGAGAGAGAGAG;

660

369. AAGGAGGAGGAGGAAAGGGAGAAGG; 5 370. GAGAGGGAGAGAGAGAGAGAGAGAG;

662

371. GAAGGGAAAGGAGGGGGAGAAGGGA; 1 372. GAGAGAGAGAGAGAGAGAGAGGAAG;

795

373. GGAGGGGAAGGGAAAAGGGAGGGGA; 1 374. GAGAGAGAGAGAGAGAGAGAGGGAG;

797

375. AAGGAGGGGAAGGGGAGAAAGGAGA; 2 376. GAGGGAGAGGGAGAGGGAGAGAGAG;

377. AGGAGGGGAAGGGGAGAAAGGAGAG; 2 378. GAGGGAGAGAGAGAGAGAGAGAGAG;

792

379. GAGGGGAGAAGAGAGGGAAGGGAGG; 2 380. GGGAGAGGGAGAGGGAGAGAGAGAG;

381. GGAAGGGGGAAGGGAGAAGAGGGAA; 2 382. GAGAGAGAGAGAGAGAGAGAGAGGG;

926

383. GGGGAAGAAGGGAGAGGAGGGAAAA; 2 384. GAGAGAGAGAGAGAGAGAGAGAGGA;

926

385. AAAGGGAGAGGGAAGGGAGAAGGGG; 3 386. GGGAGAGAGAGAGAGAGAGAGAGAG;

924

387. GAAAGGGAGAGGGAAGGGAGAAGGG; 3 388. AGAGAGAGAAGGGAAGGAGAGAGAG; 1 389. GAAGGAGGAGGAGGAAAGGGAGAAG; 5 390. GAAGAAGGGAGAGGAGAGGGAGAGA; 1 391. GGAGGGGAGGGGAAAAGGGAGAGAA; 2 392. GAAGGAAAGGGGAGAGAGAGAAGGA; 1 393. GGGAAAAGGGAGGGGAGAGGGAGAA; 2 394. GAGAAGGAAAGGGGAGAGAGAGAAG; 1 395. AAGGAGGAGGAGGAAAGGGAGGGGG; 5 396. GAGAGAAGGAAAGGGGAGAGAGAGA; 1 397. AGGGGAGAGGGGAGAAGGGGGAGGG; 3 398. GGGAGAAAGGGAGAGGGAAGGGAGA; 1 399. GAAGGGGGAGGAAGGGGAAGGAAAA; 2 400. GGGAGAAAGGGGAGGGGAAGGAGGG; 1 401. GGGAAAAGGGAGGGGAGAAGAGAGG; 2 402. GAGAGAGAGAGAGAGAGAGAGAGAG;

1056

403. GGGAGAAGAGAGGGAAGGGAGGGGA; 2 404. GAGAAGAGAAGAAGGAGGAAGGAGG;

405. GGGAGGGAGGAAGGGAGAAGGAGGG; 6 406. AGGGGGAGGGGGAGGGGAGAGGGGA; 3

407. GAAGGGAGGGGAGGGGAAGAAGGAA; 3 408. AGGGAGAGGGAGAGGGAGAGAGAGA;

409. GAGGGGAGGGGAAGAAGGAAGGGGA; 3 410. AAGGGGGAGAGAGAGAAGGGAAGGA; 1 411. AAAGGAGGGGAAGGGGAGAAAGGAG; 2 412. AAGGGGGAGAGGGGGAGGGGAAAAG; 1 413. AAGAGGGGGGAGGGGAAGAAGGGAG; 2 414. AGAAGGAAAGGGGAGAGAGAGAAGG; 1 415. AAGGGGGAGGAAGGGGAAGGAAAAG; 2 416. GAAGAAGGGAGGGAGGGGGAGAAGG; 1 417. AGGGGGAGGAAGGGGAAGGAAAAGA; 2 418. GAAGGGGGAGAGAGAGAAGGGAAGG; 1 419. GAAAGGAGGGGAAGGGGAGAAAGGA; 2 420. GGAAGGGGGAGAGAGAGAAGGGAAG; 1 421. GAGAGGGAGAGAAGAAGGGGAAGGG; 2 422. AAGGGGAGGGGAGGGAAGAAGGGGG; 1 423. GGAGAAGAGAGGGAAGGGAGGGGAG; 2 424. AGAGAGAAGGGAAGGGGAGAGAGAG; 1 425. GGGAAGGGGGAAGGGAGAAGAGGGA; 2 426. GAGAAGGGGGAGAGGGGGAGGGGAA; 1 427. GGGGAGGAAGGGGGAGGAAGGGGAA; 2 428. GGAGAGAGAGAAGGAAAGGGGAGAG; 1 429. AAGGAGGAGGAGGAAAGGAAGGGGG; 5 430. GGAGAGAGAGAAGGGAAGGAGAGAG; 1 431. GGAGGGGAGGGGAAGAAGGAAGGGG; 3 432. GGGAGAAGGGGGAGAGGGGGAGGGG; 1 433. AAGGGAAAGGAGGGGGAGAAGGGAG; 1 434. GGGAGAGAGAGAAGGAAAGGGGAGA; 1 435. AGGGGAAAAGGGGGAGGGAGAGAAG; 1 436. GGGGGAGAGAGAGAAGGGAAGGAGA; 1 437. GAGAAGGGAAAGGAGGGGGAGAAGG; 1 438. AGAAGGGGGAGAGGGGGAGGGGAAA; 1 439. GAGGGAAAAAGGGAGAAGGAGGGGG; 1 440. AGGGGGAGAGAGAGAAGGGAAGGAG; 1 441. GAGGGAGAGAAGAAGGGGAAGGGAG; 1 442. GAAAGAAGGGGAGGGGAGGGAAGAA; 1 443. GGAGAAGGGAAAGGAGGGGGAGAAG; 1 444. GAAGGGGGAGAGGGGGAGGGGAAAA; 1 445. GGGAGGGGGAGAGAAAGAAGGGGAG; 1 446. GAGAAGAGAAGGGGGAGAGAGGAGA; 1 447. GGGGGAGAAAGGAGGGGGAGGGAGA; 1 448. GGAGAAGGGGGAGAGGGGGAGGGGA; 1 449. GGGGGAGGGGGAGAAAGGAGGGGAA; 1 450. GGAGAGGGAGAGGGAGAGGGAGAGG;

451. GGGGGGAGAAAGGAGGGGGAGGGAG; 1 452. GAAAAGGGGGAGGGAGAGAAGGAGG; 1 453. AAGGAGGGAGGGGAAGAAGGGGAGG; 2 454. GAAGGGGAGGGGAGGGAAGAAGGGG; 1

455. GAGAAGGAAGGAGGGGAAGAAGGGA; 2 456. GAGGAAGAGGAGGAGGGAAAGGAAA; 1 457. GAGGGGAAGAAGGAAGGGGAGAAGG; 2 458. GGAGAAAGGGGAGGGGAAGGAGGGA; 1 459. GGAGAAGGAAGGAGGGGAAGAAGGG; 2 460. GGAGGGGAGGGGAGGAGGAGGGGAA; 2 461. GGGGAAGGGGGAAGGGAGAAGAGGG; 2 462. GGAGGGAGGGAGGAAGGGAGAAGGA; 4 463. GAGAAAGGGAGAGGGAAGGGAGAAG; 3 464. GAGAAGAGAAGAGAGAGGAGGGAGA; 2 465. GGAGGAAGGGAGGGGAGAAGGGGGA; 3 466. GAGAAGAGAAGAGGGAAGAGGGAGA; 3 467. AAGGGAGGGGAGGGGAAGAAGGAAG; 3 468. AAGGAAAGGGGAGAGAGAGAAGGAG; 1 469. AAGGAGGAGGAAGAAAGGGAGGGGG; 2 470. AGAGAGAGAAGGGGAGGGGAGAAGG; 1 471. AGGGGAAGGGGAGAAAGGAGAGGAA; 2 472. GAGAGAGAGAAGGGAAGGAGAGAGA; 1 473. AGGGGAAGGGGGAAGGGAGAAGAGG; 2 474. GGAGAAGAGAAGAGGGAAGAGGGAG; 3 475. GAAGGGGAAGGGGGAAGGGAGAAGA; 2 476. AGAGGGAGAGGGAGAGGGAGAGGGA;

477. GAGAAAGGAGGGGAAGGGGAGAAAG; 2 478. AGAGAAGAGAAGAGGGAAGAGGGAG; 3 479. GGGAGAAAGGAGGGGAAGGGGAGAA; 2 480. GAGAGGGAGAGGGAGAGGGAGAGGG;

481. AGGGAAAGGAGGGGGAGAAGGGAGA; 1 482. AGAGAAGAGGAGGGGAGGGAGGAGA; 1 483. GAAGGGGAGAAGAGGGAAGGGGAGA; 1 484. AGGAGAAGAGAGGGAGAAGGGGAGA; 1 485. GGGAAAAGGGGGAGGGAGAGAAGGA; 1 486. GGAGAAGAGAAGAGAGAGGAGGGAG; 2 487. GGGGAGAAAGGAGGGGGAGGGAGAA; 1 488. AGGGAGAGGGAGAGGGAGAGGGAGA;

489. GGGGAGGGGGAGAAAGGAGGGGAAG; 1 490. AAAGAAGGGGAGGGGAGGGAAGAAG; 1 491. AAGGGGAAGGGGGAAGGGAGAAGAG; 2 492. GAGAGAGAAGGGAAGGAGAGAGAGA; 1 493. AGAAAGGAGGGGAAGGGGAGAAAGG; 2 494. GGGAGAGGGAGAGGGAGAGGGAGAG;

495. AGAAGGGGAAGGGGGAAGGGAGAAG; 2 496. AGAAGGGGAGGGGAGGGAAGAAGGG; 1 497. GAAGGGAGGGGAGGGGGAGAAAGGG; 2 498. GAGAGAGGAGAAGAGAGGGAGAAGG; 1 499. GGAGAAAGGAGGGGAAGGGGAGAAA; 2 500. AGAGAGGGAGAGGGAGAGGGAGAGG;

501. GGGAGGAAGGGGGAGGAAGGGGAAG; 2 502. GGAGAGGGAGAGGGAGAGGGAGAGA;

503. AGAAGGGAGGGGAGGGGAAGAAGGA; 3 504. AAAGAAGGAAGGGGAGGGGAGGAAG; 1 505. AGAAAGGGAGAGGGAAGGGAGAAGG; 3 506. AAGAAGGGAGAGGAGAGGGAGAGAG; 1 507. AGAGAAGGGGAGGGGAGAAGGGGGA; 3 508. AGGAGAGAGAAGAGGGGAGGGAGGG; 1 509. AGGGAGGGGAGGGGAAGAAGGAAGG; 3 510. AGGGAGAGAGAGAAGGAAAGGGGAG; 1 511. GGAGAAGGGGGAGAGGGAGAGAAGA; 3 512. GAAAGAAGGAAGGGGAGGGGAGGAA; 1 513. GGGAGGGGAGGGGAAGAAGGAAGGG; 3 514. GGAGAGAGAAGAGGGGAGGGAGGGA; 1 515. GAGAAGAAGAGAGGGAAGGGAGGGG; 2 516. GGGGGGAGGGGAGGGGGGGGAGGGG; 1 517. GGAGGAAGGGGGAGGAAGGGGAAGG; 2 518. AAGAAGGGGAGGGGAGGGAAGAAGG; 1 519. GAGAAGGGGGAGAGGGAGAGAAGAA; 3 520. AGAGGAGAAGAGAGGGAGAAGGGGA; 1 521. GGAGAAAGGGAGAGGGAAGGGAGAA; 3 522. GAGAAGAGAAGGGAGAGGGAGGAGA; 1 523. AAGGGGAGAAGAGGGAAGGGGAGAA; 1 524. GGAAAGAAGGAAGGGGAGGGGAGGA; 1 525. AAGGGGGAAGGGAGAAGAGGGAAAG; 1 526. GAGAGAGAAGAGGGGAGGGAGGGAG; 1 527. AGAGGGAAGAGGGGGGAGGGGAAGA; 1 528. GAGAGAGAGAAGGGGAGGGGAGAAG; 1 529. GAAGGGAGAGGAGGGGGAGAAAGGA; 1 530. GAGAGGAGAAGAGAGGGAGAAGGGG; 1 531. GAGGGGAAAAGGGAGAGAAGAAGAG; 1 532. GAGGAGAAGAGAGGGAGAAGGGGAG; 1 533. GGGAGAGGAGGGGGAGAAAGGAGAG; 1 534. AGAGAGAGGGAGAGGGAGAGGGAGA;

535. GGGGAAAAGGGGGAGGGAGAGAAGG; 1 536. GAGAGAGGGAGAGGGAGAGGGAGAG;

537. GGGGGGAGGGGGAGAAAGGAGGGGA; 1 538. AAGAAGGAAGGGGAGGGGAGGAAGG; 1 539. AGAGAAGAAGAGAGGGAAGGGAGGG; 2 540. AGAGAGGAGAAGAGAGGGAGAAGGG; 1 541. AGGGGGAGGAAGGGGGAGGAAGGGG; 2 542. GAGAAAGGGGAGGGGAAGGAGGGAG; 1 543. GAAGAAGAGAGGGAAGGGAGGGGAG; 2 544. GAGAGAAGGGAAGGGGAGAGAGAGA; 1

545. GAAGGGGGAGGAAGGGGGAGGAAGG; 2 546. GAGGAGGGGGAGGAGGGGGAGGGGA; 2 547. GAGGAAGGGGGAGGAAGGGGAAGGA; 2 548. GAGAGGGAGAGGGAGAGGGAGAGAG;

549. GGAGAGGGAGAGAAGAAGGGGAAGG; 2 550. AGAGGGAGAGGGAGAGGGAGAGAGA;

551. GGAGGAAGGGGGAGGAAGGGGGAGG; 2 552. AGAAGGAAGGGGAGGGGAGGAAGGG; 1 553. GGAGGGGAAGAAGGAAGGGGAGAAG; 2 554. GAAGGAAGGGGAGGGGAGGAAGGGA; 1 555. GGGGAAGAAGGGAGAGGAGAGGGAG; 2 556. GGAGAGGAGAAGAGGGAGGAGGGAG;

557. AGAAGGGGGAGAGGGAGAGAAGAAG; 3 558. AAGGGGAGGGGAGGAAGGGAGGGGA; 1 559. AAGGGGGAGGAAGGGGGAGGAAGGG; 2 560. GGGAGGGGAGGGGAGGAGGAGGGGA; 2 561. AGGAGGGAGGGGAAGAAGGGGAGGG; 2 562. GAGGGAGGGGAGGGGAGGAAGGAGG; 6 563. GAGAAGAGAGGGAAGGGAGGGGAGG; 2 564. AGAGAAGGGAAGGGGAGAGAGAGAA; 1 565. GAGAGAAGAAGAGAGGGAAGGGAGG; 2 566. AGGAAAGGGGAGAGAGAGAAGGAGA; 1 567. GGAGAGAAGAAGAGAGGGAAGGGAG; 2 568. GAAGGGGAGGGGAGGAAGGGAGGGG; 1 569. GAAAGAGAGAGAGAGAGGGAGGGAG;

570. GAGAGAGAGAAGGAGAGGAGAGAAG; 1

571. AGGGAGAGAAGAAGGGGAAGGGAGA; 1 572. AGAGGGAGGGGAGGGGAGGAAGGAG; 6 573. GAAAGGAGGGGGAGAAGGGAGAGGA; 1 574. GGAGAGGGAGAGGGAGAGAGAGAGA;

575. GAGAGGGAAGAGGGGGGAGGGGAAG; 1 576. AAAAGGGGGAGGGAGAGAAGGAGGG; 1 577. GAGAGGGAAGGGAGAAGGGGGGAAA; 1 578. AGAGAGAGGAGAAGAGAGGGAGAAG; 1 579. GAGGGGGAGAGAAAGAAGGGGAGGG; 1 580. GGAGGGGAGGAAGGGAGGGGAGAAG; 1 581. GGAAAGGAGGGGGAGAAGGGAGAGG; 1 582. GGGGAGGGGAGGAAGGGAGGGGAGA; 1 583. GGAGAGGAGGGGGAGAAAGGAGAGG; 1 584. GAGAGAGAGAGAGAAGGGGAGGGGA; 3 585. GGGAAAGGAGGGGGAGAAGGGAGAG; 1 586. GAGAAGAGAAGAGGGAGGGAGGAGA;

587. GGGAGAAAGGAGGGGGAGGGAGAAG; 1 588. AGAGAAGAGAAGAGGGAGGGAGGAG;

589. AGAGGGGGGAGGGGAAGAAGGGAGG; 2 590. AGAAGGGAGAGGAGAGGGAGAGAGA; 1 591. GAGGAAGGGGGAGGAAGGGGGAGGA; 2 592. AGAGAGAAGAGGGGAGGGAGGGAGG; 1 593. GAGGGAGGGGAAGAAGGGGAGGGGG; 2 594. AGAGGAAGAGGAGGAGGGAAAGGAA; 1 595. AAGGGGGAGAGGGAGAGAAGAAGGG; 3 596. AGGAGAAGAGAAGAGGGAGGAGGGA;

597. GGGGAAAGAAAGGGAGGAAGGAAGG; 5 598. GAGAAGAGAAGAGGGAGGAGGGAGA;

42 599. GAAGGGAAGGAGGGAGGAGGGAGGA;

600. AGGGAGAGGAGAGGGAGAGAGAGAG; 3

601. AAGAAGAGAGGGAAGGGAGGGGAGG; 2 602. AGAGAGAGAGAGGGAGAGGGAGAGG;

603. AGAAGAAGAGAGGGAAGGGAGGGGA; 2 604. AGAGAGGAGAAGAGAAGAGGGAGGA;

605. AGAAGGGAGGGGAGGGGGAGAAAGG; 2 606. AGAGAAGAGAAGGGAGAGGGAGGAG; 1 607. GAGGGGAGAGGGGAGAAGGGGGAGG; 2 608. GGGAGGGAGGAAGAGAGAAGAAGGG; 1 609. GGAGGGAGGGGAAGAAGGGGAGGGG; 2 610. GGGAGGGGAGGAAGGGAGGGGAGAA; 1 611. GGGAGGGGAAGAAGGAAGGGGAGAA; 2 612. GGAGAAGAGAAGAGGGAGGAGGGAG;

613. GGGAGGGGAAGAAGGGGAGGGGGAG; 2 614. GAGAGGAGAAGAGAAGAGGGAGGAG;

615. GGGGGAGGAAGGGGGAGGAAGGGGA; 2 616. AAGGAGAAGAGAAGAGGGAGGAGGG;

617. AAAGGAGGGGGAGAAGGGAGAGGAA; 1 618. GAGGAGAAGAGAAGAGGGAGGAGGG;

619. AGGGGAGGGAAGAAGGGGGAGGGGG; 1 620. AAGGAAGGGGAGGGGAGGAAGGGAG; 1 621. GAGAAGGGAAGAAGAAGGAGGAGAG; 1 622. GAGAAGGGAAGGGGAGAGAGAGAAG; 1 623. GAGAGGAGGGGGAGAAAGGAGAGGA; 1 624. GGAAGGGGAGGGGAGGAAGGGAGGG; 1 625. GGAAGAAGGGAGAGGAGGGAAAAGG; 1 626. AGAGGAGAAGAGAAGAGGGAGGAGG;

627. GGAGGGAAAAGGGGAAGGAGGGGAA; 1 628. GGGAGGGAGGGAGGAAGGGAGAAGG; 4 629. GGAGGGGGAGAAAGGAGGGGAAGGG; 1 630. GGGAAGGGAAGGAGGGAGGAGGGAG; 2 631. GGAGGGGGAGAGAAAGAAGGGGAGG; 1 632. AGGGGAGGGGAGGAAGGGAGGGGAG; 1 633. GGGAGAAGGGAAGAAGAAGGAGGAG; 1 634. GAAAGGGGAGGGGAAGGAGGGAGGG; 1 635. GGGAGGGGGAGAAAGGAGGGGAAGG; 1 636. GAGGGAGAGAGAGAAGGAAAGGGGA; 1 637. GGGGAGAAGGGAAGAAGAAGGAGGA; 1 638. AGAGAGAAGAGAAGAGGGAGGAAGG;

639. AGAGGGAGAGAAGAAGGGAAAGGGA; 2 640. GAAGAGAAGAGAAGAGGGAGGAAGG;

641. AGGGAGGGGAAGAAGGGGAGGGGGA; 2 642. GAGAGAAGAGAAGAGGGAGGAAGGA;

643. GAGGGGAGGGGGAGAAGGGAGGGGA; 2 644. GGAGAAGAGAAGAGGGAGGAAGGAG;

645. GAAGGGGGAGAGGGAGAGAAGAAGG; 3 646. AGAAAGGGGAGGGGAAGGAGGGAGG; 1 647. AGGAGGAGAAGGGGGAGGGGGGAGA; 4 648. GAGAAGAGAAGAGGGAGGAAGGAGA;

655. AGAGAGAAGGGGAGGGGAGAAGGGG; 2 656. GGAGAGAGAGAGAGAAGGGGAGGGG; 3 657. AGAGGAGAGAAGGGGGAGGGGGAGG; 2 658. AGAGAAGAGAAGGGGGAGAGAGGAG; 8 659. AGGGGAGGGGAAGAAGGAAGGGGAG; 2 660. GAGAAGAGAAGGGGGAGGGAGGAGA;

661. GGGAGGAGAGAGGAGAGGGAGGAGA; 2 662. GAAGGGAGAGGAGAGGGAGAGAGAG; 1 663. GGGGAGGGGAAGAAGGAAGGGGAGA; 2 664. AGAGAAGAGAAGGGGGAGGGAGGAG;

665. GAGGAGGAGAAGGGGGAGGGGGGAG; 4 666. AGAGGGAGAGGGAGAGAGAGAGAGA;

667. GGGAAAGAAAGGGAGGAAGGAAGGA; 5 668. AAAGGGGAGGGGAAGGAGGGAGGGA; 1 669. AAGGGAGAGGAGGGGGAGAAAGGAG; 1 670. AGAGGGAGAGAGAGAAGGAAAGGGG; 1 671. AGGGAGAGGAGGGGGAGAAAGGAGA; 1 672. GAGAGAAGAGGGGAGGGAGGGAGGA; 1 673. AGGGGGAAGGGAGAAGAGGGAAAGG; 1 674. GGAAAGGGGAGAGAGAGAAGGAGAG; 1 675. GAAGAAGGGAGAGGAGGGAAAAGGA; 1 676. GAGAGGAAGAGGAGGAGGGAAAGGA; 1 677. GAGGGAAGAGGGGGGAGGGGAAGAA; 1 678. GGAAGGGAAGGAGGGAGGAGGGAGG; 2 679. GAGGGGGAGAAAGGAGGGGAAGGGG; 1 680. AAGGAGAGAGAGAGAGAAGGGGAGG; 4 681. GGAAGGGGAGAAGAGGGAAGGGGAG; 1 682. AGAGAAGAGAAGGGGGAGGAAGGAG; 8 683. GGAGAAGGGAAGAAGAAGGAGGAGA; 1 684. GGGGAGGGGAGGGGAGGAGGAGGGG; 2 685. GGAGAGGGAAGGGAGAAGGGGGGAA; 1 686. GAAAAGAGAAGGGGGAGGGAGGAGA; 8 687. GGAGGGGAAAAGGGAGAGAAGAAGA; 1 688. GAGAAAGGAAGGGGGAGGGAGGAGA; 7 689. GGGAGAGAAAGAAGGGGAGGGGAGG; 1 690. AAAGGGGAGAGAGAGAAGGAGAGGA; 1 691. GGGAGGGGAAAAGGGAGAGAAGAAG; 1 692. AGAGAGAAGGGAAGGAGAGAGAGAG; 1 693. GGGGAGAGAAAGAAGGGGAGGGGAG; 1 694. AGAGGGAGGGGAGGGGAGGGAGGAG; 6 695. GGGGAGGGAAGAAGGGGGAGGGGGA; 1 696. GAGGGAGGGGAGGGGAGGGAGGAGG; 6 697. GGGGGAGAGAAAGAAGGGGAGGGGA; 1 698. GAGGGAGAGGGAGAGAGAGAGAGAG;

699. GAGAGGAGAGAAGGGGGAGGGGGAG; 2 700. AGAGAGGAAGAGGAGGAGGGAAAGG; 1 701. GGGAAGAAGGGAGAGGAGAGGGAGA; 2 702. GGAGAGAGAGAAGGAGAGGAGAGAA; 1 703. GGGGAGAGGGAGAGAAGAAGGGGAA; 2 704. GAGAAGAGAAGGGGGAGGAAGGAGA; 7 705. GGAAGGGAGGGGAGGGGAAGAAGGA; 3 706. AGGGAGAAAGGGAGGGAGGAAGGAA; 2 707. GGAGGAAGGAGGGAGGAGGGAGGAG;

711. GAGAGAGAGGGAGGGAGGGAGGGAG;

712. GGAAGAAGGGAGGGAGGGGGAGAAG; 1

713. GGAAGGGAGGGGAGGGGAAGAAGGG; 3 714. GGGAAAAGAGGGAGGAGGAGAAGGG; 1 715. AGGAAGGAGGGAGGAGGGAGGAGGG;

1074

716. GGGAGAGAGAGAAGGAGAGGAGAGA; 1

717. AGAAGAGAGGGAAGGGAGGGGAGGG; 2 718. AGGAGAGAGAGAGAGAAGGGGAGGG; 3 719. GAGGGGGGAGGGGAAGAAGGGAGGG; 2 720. AAGAGGGAGAGAGAGAAGGAAAGGG; 1 721. GGAGGAGAGAGGAGAGGGAGGAGAG; 2 722. AAGGGGAGAGAGAGAAGGAGAGGAG; 1 723. GGGGGAGAGGGAGAGAAGAAGGGGA; 2 724. AGGGAGGGAGGAAGAGAGAAGAAGG; 1 725. GGAGGAGGAGAAGGGGGAGGGGGGA; 4 726. GAAAGGGGAGAGAGAGAAGGAGAGG; 1 727. GAGGGAGGGAGGGAGGGAGAGAGAG;

728. GGGGAGAGAGAGAAGGAGAGGAGAG; 1

729. AAAAGGGAGGGAAGAGGGGGAAAAG; 1 730. GAGAGGAGAGGGAGGAGAGAGGAGG; 3 731. AAAGAAGGGAGAGGAGGGGAAGAAG; 1 732. AGAAAAGAGAAGGGGGAGGGAGGAG; 7 733. AGGGGAGGGGGAGAAAGGGAGAGGG; 1 734. AGAGAAGAGGGGAGGGAGGGAGGAA; 1 735. GAAAGAAGGGAGAGGAGGGGAAGAA; 1 736. AGAGGAGAGGGAGGAGAGAGGAGGG; 3 737. GGAAAAGGGGGAGGGAGAGAAGGAG; 1 738. AGGGAGGGAGGGAGGAAGGGAGAAG; 4 739. GGGAAGAGGGGGGAGGGGAAGAAGG; 1 740. AGGGGGGGAGGGGGAGGGGGGAGGG; 1 741. GGGGAAAAGGGAGAGAAGAAGAGAG; 1 742. GAAGAGGGAGAGAGAGAAGGAAAGG; 1 743. GGGGAGGGGAAAAGGGAGAGAAGAA; 1 744. GAGAGAAAGAGAGGGAGGAGGAAAG; 1 745. GGGAGAGGGAGAGAAGAAGGGGAAG; 2 746. GAGGGGGGGAGGGGGAGGGGGGAGG; 1 747. GAGAAAGAGAGAGAGAGAGGGAGGG;

748. AAGGAGGAGGAGAAGGAGAAAGGGG; 1

749. AGAGAGGGAAGGGAGGGGAGGGGAA; 3 750. GGGAGGGGAGGGGAGGAAGGAGGGA; 1 751. GGAGGGAGGGAGGGAGGGAGAGAGA;

755. AGAGGGAAGGGAGGGGAGGGGAAGA; 3 756. GAAGAAGGAGGAAGGAGAGAGAAGG; 4

757. GAGAGGGAAGGGAGGGGAGGGGAAG; 3 758. AGGGAGGGGAGGGGAGGGAGGAGGG; 5 759. AAGAAGGGAGAGGAGGGAAAAGGAG; 1 760. GAAGGAGAGAGAGAGAGAAGGGGAG; 4 761. AAGAAGGGAGAGGAGGGGAAGAAGG; 1 762. GGAAGGAGAGAGAGAGAGAAGGGGA; 4 763. AAGGGAGGGGAGGGGGAGAAAGGGA; 1 764. GAGAGAAGGGAAGGAGAGAGAGAGA; 1 765. AGAAGGGAGAGGAGGGAAAAGGAGA; 1 766. GGAAGAGGGAGAGAGAGAAGGAAAG; 1 767. AGGGGAGGGGAAAAGAGGGAGGAGG; 1 768. GGGGGGGAGGGGGAGGGGGGAGGGG; 1 769. AGGGGGAGAAAGGAGGGGAAGGGGA; 1 770. AGAGAGAGAGAGGGAGGGAGGGAGG; 6 771. AGGGGGAGAGAAAGAAGGGGAGGGG; 1 772. AGGGGAGGGGAAGGAGGGAGGGAAA; 1 773. GAAGAGGGGAGAAAGGGGAGGGGAA; 1 774. GAGAGAGAGAGAGGGAGGGAGGGAG; 6 775. GAGGGAAGAAGGGGGAGGGGGAGAA; 1 776. AAAGAGAGAGAGAGAGGGAGGGAGG; 8 777. GAGGGGAGGGGGAGAAAGGGAGAGG; 1 778. GGGAAGAAGGGAGGGAGGGGGAGAA; 1 779. GGAAAAGGGAGAGAAGAAGAGAGGG; 1 780. AAGGGAGAGGAGAGGGAGAGAGAGA; 1 781. GGAAGAGGGGGGAGGGGAAGAAGGG; 1 782. GAGAAGAGGGGAGGGAGGGAGGAAG; 1 783. GGAGGGGAGGGAAGAAGGGGGAGGG; 1 784. AAGAGAGAGAGAGAGGGAGGGAGGG; 8 785. GGGAAAAGGGAGAGAAGAAGAGAGG; 1 786. GAGAGAGAGAGGGAGGGAGGGAGGG; 6 787. GGGAGGGAAGAAGGGGGAGGGGGAG; 1 788. AGGGAGGGGAGGGGAGGAAGGAGGG; 1 789. GAGAGAGGAGAGGGAGGAGAGAGGA; 2 790. AGGGAGAGGGAGAGAGAGAGAGAGA;

791. GAGAGGGAGAGAAGAAGGGAAAGGG; 2 792. GGAGGGGGGAGGGGGGAGGGGAAGA; 1 793. AGAGAAGAAAAGGGGGAGGGAGGAG; 3 794. AGAGAGAGAGAGAGGGAGGGAGGGA; 8 795. GAGGGAAGGGAGGGGAGGGGAAGAA; 3 796. AGAGAGAGAGAGAGGGAGAGGGAGA; 8 797. GGGAAGGGAGGGGAGGGGAAGAAGG; 3 798. AGGGAGAAGGGAGGAGGGAGGAAGG; 6 799. AGGGAAGGAGGGAGGAGGGAGGAGG;

811. AGAGAGGAGAGGGAGGAGAGAGGAG; 2 812. GGGAGGGAGGGAGGGAGAGAGAGAG; 6 813. AGGGGGAGAGGGAGAGAAGAAGGGG; 2 814. GGGAGGGGAAGGAGGGAGGGAAAGA; 1 815. GAGAGAGAGAGAGAGAGGGAGAGGG;

819. AGAAGAGGGGAGAAAGGGGAGGGGA; 1 820. GGAGAGGGAGAGAGAGAGAAGGGGA; 5 821. AGGGAAGAAGGGGGAGGGGGAGAAA; 1 822. GAGGGGGAGGGGGGAGGGGGGAGGG; 1 823. AGGGAAGAGGGGGGAGGGGAAGAAG; 1 824. GGGAGGGGGAGGGGGGAGGGGGGAG; 1 825. AGGGGGAGAAGGGGGAGGAGAGAAG; 1 826. GAGGGAGGGAGGGAGAGAGAGAGAG; 6 827. GAAGAGAGGGAGAAGGGGAGAGGAA; 1 828. GGGAGAAAGGGAGGGAGGAAGGAAG; 2 829. GAGAGAAGGGGAAGGGGGAAGGGAG; 1 830. AGAAGAGGGGAAGGGAGGGAGGGAG; 1 831. GAGGGGAGAAGGGGGAGAGGGGGAG; 1 832. GGGAGGGGGGAGGGGGGAGGGGAAG; 1 833. GAGGGGAGGGAAGAAGGGGGAGGGG; 1 834. GAGAAAGGGAGGGAGGAAGGAAGGG; 2 835. GGAAAAAGGGGAGAGGAAGGGGAGA; 1 836. GGAGAAAGGGAGGGAGGAAGGAAGG; 2 837. GGAAAGAAGGGAGAGGAGGGGAAGA; 1 838. AAAGGGAGGGAGGAAGGAAGGGAGG; 2 839. GGAGAAGAGGGGAGAAAGGGGAGGG; 1 840. AGAAAGGGAGGGAGGAAGGAAGGGA; 2 841. GGAGGGGAGGGGGAGAAAGGGAGAG; 1 842. GAAAGGGAGGGAGGAAGGAAGGGAG; 2 843. GGGAAGAAGGGGGAGGGGGAGAAAG; 1 844. GGAGGGGGAGGGGGGAGGGGGGAGG; 1 845. GGGAGGGGAAAAGAGGGAGGAGGAG; 1 846. GAGGGAGGGAGAGAGAGAGAGAGAG;

847. GGGGAGGGGAAAAGAGGGAGGAGGA; 1 848. GGAGGGAGGGAGGAAGAGAGAAGAA; 1

849. GAGAGAAGGGGAGGGGAGAAGGGGG; 2 850. GAGAGAGAGAGAGAGGGAGAGGGAG; 8 851. GAGGAGAGAGGAGAGGGAGGAGAGA; 2 852. GGGGAGGGGGGAGGGGGGAGGGGAA; 1 853. AGGGAAGGGAGGGGAGGGGAAGAAG; 3 854. GGGGGAGGGGGAGGGGGGAGGGGGG; 1 855. GAGGGAGGAGGGAGGAGGGAGGAGG;

859. AAGGGAGGAGAGAGGAGAGGGAGGA; 2 860. GGGGGGAGGGGGAGGGGGGAGGGGG; 1 861. AGGGGGAGGGGAGAGGGGAAGGAAG; 2 862. AGGGAAGGAGAGAGAGAGAGAAGGG; 3 863. GGAGAGGGAGAGAAGAAGGGAAAGG; 2 864. GGGAAGGAGAGAGAGAGAGAAGGGG; 3 865. AGGGAGGGAGGGAGGGAGGGAGAGA;

869. AAGAGGGGAGAAAGGGGAGGGGAAG; 1 870. AGAGAGAGAGAGAGAGGGAGAGGGA; 8 871. AAGGGAGAAGAGGGGAGAAAGGGGA; 1 872. AGGGGGAGGGGGGAGGGGGGAGGGG; 1 873. AAGGGGAGGGGGGAGAAAGAAAGAA; 1 874. GGAGGAAGGAAGGAAAGGAAGGAAG; 1 875. GAAAAAGGGGAGAGGAAGGGGAGAA; 1 876. GGGGGAGGGGGGAGGGGGGAGGGGA; 1 877. GAGGGGGAAGGGAGAGGGGGGGAAA; 1 878. AAGGGAAGGAGAGAGAGAGAGAAGG; 2 879. GGAGGGGAAGGGGAGAAAGGAGAGG; 1 880. AGGGGAGGGGAGGGGAGGAGGAGGG; 2 881. GGGAGAAGAGGGGAGAAAGGGGAGG; 1 882. GGGAGGAAGGAAGGAAAGGAAGGAA; 1 883. AAAGGGAGGAGAGAGGAGAGGGAGG; 2 884. GGGAGAGGAGAGGGAGAGAGAGAGA; 3 885. AGGAGAGAGGAGAGGGAGGAGAGAG; 2 886. AGAAGAGGGGAGGGAGGGAGGAAGA; 1 887. AGGGAGGAGAGAGGAGAGGGAGGAG; 2 888. GGGAGGGAGGGAGGAAGAGAGAAGA; 1 889. GAGGAGAGAAGGGGGAGGGGGAGGG; 2 890. GGGAGGGAGAAGGGAGGAGGGAGGA; 2 891. GGAGAGAGGAGAGGGAGGAGAGAGG; 2 892. GGAGGGAGAAGGGAGGAGGGAGGAA; 2 893. GGGAGGAAGGGAGGGGAGAAGGGGG; 2 894. GAGGGGAGGGGAGGGGAGGAGGAGG; 2 895. AGAGAGGGAGGGAGGGAGGGAGGGA; 896. GGAGAGGAGAGGGAGAGAGAGAGAA; 3

903. GAAAGGGAGGAGAGAGGAGAGGGAG; 2 904. AGAGGAGAGGGAGAGAGAGAGAAGG; 3 905. GGGGAAAGAGAGAGAAAAGGGGAAG; 2 906. GAGAGGAGAGGGAGAGAGAGAGAAG; 3 907. AAGAGAGGGAGAAGGGGAGAGGAAG; 1 908. GGAGGGGAGGGGAGGGGAGGAGGAG; 2 909. AAGGGAGGGGAGAAGAGAGGGAAGG; 1 910. AGGGAGGAAGGAAGGAAAGGAAGGA; 1 911. AGGGAGAAGAGGGGAGAAAGGGGAG; 1 912. GGGGAGGGAGGGAGGAAGAGAGAAG; 1 913. AGGGAGGGGAGAAGAGAGGGAAGGG; 1 914. AAGAGGGGAGGGAGGGAGGAAGAGA; 1 915. AGGGGAGAAGGGGGAGAGGGGGAGG; 1 916. GGGGAGGGAGAAGGGAGGAGGGAGG; 2 917. AGGGGGGAGAAGGGGAGGGGGGAGA; 1 918. GAGAAGGGAAGGAGAGAGAGAGAGA; 1 919. GAGAAGAGGGGAGAAAGGGGAGGGG; 1 920. AGGGGAGGGAGGGAGGAAGAGAGAA; 1 921. GGAGGGAAGAAGGGGGAGGGGGAGA; 1 922. GAGGGGAGGGAGGGAGGAAGAGAGA; 1 923. GGGGAGAAAGGAGGGGAAGGGGAGA; 1 924. AGAGGGGAGGGAGGGAGGAAGAGAG; 1 925. GGGGAGGGGGAGAAAGGGAGAGGGA; 1 926. GAGAGGGAGAGGGAGAGAGAGAGAG; 3 927. GGGGGAGAAAGGAGGGGAAGGGGAG; 1 928. GGGAGGGGAGGGGAGGGGAGGAGGA; 2 929. GGGGAGAGGGAGAGAAGAAGGGAAA; 2 930. AAGGGAGGAAGGAAGGAAAGGAAGG; 1 931. GGGGAGGAAGGGAGGGGAGAAGGGG; 2 932. GGAAAAGAGGGAGGAGGAGAAGGGG; 1 933. GAGGGGGAGGGGGAGGGGAGAGGGG; 7 934. GGGGAGGGGAGGGGAGGGGAGGAGG; 2 935. GAGAGGGAGGGAGGGAGGGAGGGAG;

100

943. AGGGGAGGAAGGGAGGGGAGAAGGG; 2 944. AAAAGAGGGAGGAGGAGAAGGGGGA; 1 945. AGGGGGAGAGGGAGAGAAGAAGGGA; 2 946. AAGAGGGGAAGGGAGGGAGGGAGGA; 1 947. GGGAGAGGGAGAGAAGAAGGGAAAG; 2 948. AGGGAGGAGGAGAAGGGGGAGGGGG; 1 949. AGGGAGGGAGGGAGGGAGGGAGGGA;

950. GAGGGAGGAGGAGAAGGGGGAGGGG; 1

951. AAAAAGGGGAGAGGAAGGGGAGAAG; 1 952. AGAGGGAGGAGGAGAAGGGGGAGGG; 1 953. AAAGGGAGGGGAGAAGAGAGGGAAG; 1 954. AAGAGGGAGGAGGAGAAGGGGGAGG; 1 955. AGAAGAGAGGGAGAAGGGGAGAGGA; 1 956. GAGAGAGAGAGAGGGAGAGGGAGAG; 2 957. AGAGAAGGGGAAGGGGGAAGGGAGA; 1 958. AGAGGGGAAGGGAGGGAGGGAGGAA; 1 959. AGGGAGGGGAGGGGGAGAAAGGGAG; 1 960. GAGGGAGAAGGGAGGAGGGAGGAAG; 1 961. AGGGGAGAAAGGGGAGGGGAAGGAG; 1 962. AAGGGAGGGAGGGAGGAAGGGAGAA; 1 963. GAGAAAGGGAGGGGGAAGGGAGAGG; 1 964. GAAGGGAGGGAGGGAGGAAGGGAGA; 1 965. GAGAAGGGAGAAGAGGGGAGAAAGG; 1 966. GAGGGGAAGGGAGGGAGGGAGGAAG; 1 967. GGAAAAGGGAGGGGAGAAGAGAGGG; 1 968. AGGGGAAGGGAGGGAGGGAGGAAGG; 1 969. GGAAAGGGGAGAAGGGGAAGGGAGA; 1 970. GGGGAAGGGAGGGAGGGAGGAAGGG; 1 971. GGGAGGGGAGGGGGAGAAAGGGAGA; 1 972. GGGAAGGGAGGGAGGGAGGAAGGGA; 1

973. 974. GGAAGGGAGGGAGGGAGGAAGGGAG; 1

101

Table 6: triple target sequences and off target ratio: 20 sequences of those found by Triplexator are listed, and for each of them was calculated number of hits on the Y-chromosome, the possible off-targets and the ratio between the two values.

TTS Hits Off-targets

Ratio Hits/off targets

AAAGGAGGAGAAGGAAAAGGAGGGG 142 42 1,76

AGGGGAAGGAGAGAGAAGAGGGGGA 70 24 1,54

Im Dokument Detection of Y-chromosome bearing bovine sperm using laser-generated gold nanoparticle bio-conjugates (Seite 67-0)