Myeloid Leukemia

More documents

Recommendations

Info

Real-Time RT-PCR Strategies 33 2.1.1.2. LIGHT-UP PROBE Some reporters are intercalating asymmetric cyanine dyes that exhibit a large increase in fluorescence intensity upon binding to DNA: oxazole yellow (YO), thiazole orange, and 4-[(3-methyl-6-(benzothiazol-2-yl)-2,3-dihydro-(benzo- 1,3-thiazole)-2-methylidene)]-1-methyl-pyridinium iodide (BEBO) (8,9). The light-up probe is a peptide nucleic acid (PNA) that consists of a sequencerecognizing element linked to a single fluorescent dye that serves as a group reporter. The PNA is an uncharged nucleic acid analog to which an asymmetric cyanine dye (thiazole orange) is tethered (10). The light-up probe is flexible, allowing the dye to interact with the target nucleic acid upon hybridization. PNA are uncharged analogs to minimize internal complex formation. Upon probe hybridization, PNA binds specifically to the target nucleic acid, bringing the dye to it, which results in a large enhancement in dye fluorescence (10,11). 2.1.1.3. AMPLIFLUOR HAIRPIN PRIMERS Amplifluor hairpin primers (Amplifluor Technology) are designed so that a fluorescent signal is generated only when the primer is unfolded during its incorporation into an amplification product (12). The fluorescence signal produced directly correlates with the accumulation of PCR product at each cycle. Each Amplifluor primer consists of a 3� 18-base oligonucleotide tail (Z sequence) and a 5� intracomplementary sequence labeled with paired energytransfer molecules (the fluophore and the quencher DABSYL). The Z sequence acts as a universal PCR primer and is specifically designed to reduce PCR background due to heterodimer formation. The Z sequence is added to the 5� end of one of the target-specific primers. At the annealing temperature, the Z sequence anneals to the complementary sequence contained in an amplicon generated in the initial cycles of the reaction. The hairpin is unfolded, allowing an increase in fluorescence emission (Fig. 3). Light upon extension (LUX) primers (Invitrogen) are a variation on hairpin primers. They are oligonucleotides of 20–30 bases labeled with a single fluorophore (FAM or JOE) close to the 5� end in a hairpin structure (13). This fluorogenic primer has a short sequence tail of 4–6 nucleotides on the 3� end that is complementary to the 5� end of the primer (Fig. 4). This configuration intrinsically renders fluorescence quenching capability, so that a separate quenching moiety is not needed. When the primer is incorporated into the double-stranded PCR product, the PCR LUX primer is dequenched, resulting in a significant increase in fluorescence signal. The fluorogenic primer may be the forward or the reverse primer. The characteristics of the primers, such as length and Tm, are included in the primer design by proprietary software, called LUX Designer (Invitrogen, http://www.invitrogen.com/lux). These design rules enable the software to output numerous primer pairs that are located throughout the target (input) sequence.
34 Picard, Silvy, and Gabert Fig. 3. Double-stranded DNA detection with Amplifluor hairpin primers. One of the target-specific primers possesses at its 5� end the Z sequence, which acts as a universal polymerase chain reaction primer. During the initial cycle of the reaction, an amplicon is generated that contains the complement sequence to the Z sequence (Z� sequence). The Z� sequence specifically anneals to a hairpin primer. This hairpin amplifluor primer consists of a 3� oligonucleotide tail (Z sequence) and a 5� intracomplementary sequence labeled with a pair of energy-transfer molecules. As the hairpin amplifluor primer is incorporated, the hairpin is unfolded; quenching is no longer possible as a result of increased distance between the reporter (circle) and the quencher (pentagon).
Page 1 and 2: M E T H O D S I N M O L E C U L A R
Page 3 and 4: M E T H O D S I N M O L E C U L A R
Page 5 and 6: © 2006 Humana Press Inc. 999 River
Page 7 and 8: vi Preface comprehensive review of
Page 9 and 10: viii Contents 11 Detection of the F
Page 11 and 12: x Contributors D. GARY GILLILAND
Page 14 and 15: RNA and DNA From Leukocytes 1 1 Iso
Page 16 and 17: RNA and DNA From Leukocytes 3 mine
Page 18 and 19: RNA and DNA From Leukocytes 5 late
Page 20 and 21: RNA and DNA From Leukocytes 7 9. Us
Page 22 and 23: RNA and DNA From Leukocytes 9 16. C
Page 24 and 25: RNA and DNA From Leukocytes 11 3. I
Page 26 and 27: Cytogenetic and FISH Techniques 13
Page 40 and 41: Real-Time RT-PCR Strategies 27 3 Ov
Page 42 and 43: Real-Time RT-PCR Strategies 29
Page 44 and 45: Real-Time RT-PCR Strategies 31 cifi
Page 48 and 49: Real-Time RT-PCR Strategies 35 Fig.
Page 50 and 51: Real-Time RT-PCR Strategies 37 2.1.
Page 52 and 53: Real-Time RT-PCR Strategies 39 the
Page 56 and 57: Real-Time RT-PCR Strategies 43 duri
Page 58 and 59: MyiQ single color 400 nm 585 nm 96
Page 60 and 61: Real-Time RT-PCR Strategies 47 side
Page 62 and 63: Table 2 Real-Time Quantitative Poly
Page 64 and 65: Real-Time RT-PCR Strategies 51 AML-
Page 66 and 67: Real-Time RT-PCR Strategies 53 the
Page 68 and 69: Real-Time RT-PCR Strategies 55 plas
Page 70 and 71: Real-Time RT-PCR Strategies 57 betw
Page 72 and 73: Real-Time RT-PCR Strategies 59 Ct C
Page 76 and 77: Real-Time RT-PCR Strategies 63 asse
Page 78 and 79: Real-Time RT-PCR Strategies 65 21.
Page 80 and 81: Real-Time RT-PCR Strategies 67 50.
Page 82 and 83: Quantitative PCR for Monitoring CML
Page 96 and 97:
Quantitative PCR for Monitoring CML
Page 98 and 99:
Page 100 and 101:
Page 102 and 103:
Page 104 and 105:
Page 106 and 107:
Detection of BCR-ABL Mutations 93 5
Page 108 and 109:
Detection of BCR-ABL Mutations 95 F
Page 110 and 111:
Detection of BCR-ABL Mutations 97 2
Page 112 and 113:
Detection of BCR-ABL Mutations 99 o
Page 114 and 115:
Detection of BCR-ABL Mutations 101
Page 116 and 117:
Page 118 and 119:
Page 120 and 121:
Deletion of Derivative Chromosome 9
Page 122 and 123:
Page 124 and 125:
Page 126 and 127:
Page 128 and 129:
Diagnosis of PML-RARA-Positive APL
Page 130 and 131:
Page 132 and 133:
Page 134 and 135:
Page 136 and 137:
Page 138 and 139:
Page 140 and 141:
Duplexed QZyme RT-PCR for APL Analy
Page 142 and 143:
Page 144 and 145:
Page 146 and 147:
Page 148 and 149:
Page 150 and 151:
Page 152 and 153:
Page 154 and 155:
Page 156 and 157:
Page 158 and 159:
Page 160 and 161:
Page 162 and 163:
Diagnosis of AML1-MTG8 (ETO)-Positi
Page 164 and 165:
Page 166 and 167:
Page 168 and 169:
Page 170 and 171:
Page 172 and 173:
Page 174 and 175:
Page 176 and 177:
Diagnosis of CBFB-MYH11-Positive AM
Page 178 and 179:
165 Table 1 Primers for CBFB-MYH11
Page 180 and 181:
Page 182 and 183:
Page 184 and 185:
Page 186 and 187:
Page 188 and 189:
Page 190 and 191:
FIP1L1-PDGFRA in Hypereosinophilic
Page 192 and 193:
Page 194 and 195:
Page 196 and 197:
Page 198 and 199:
Page 200 and 201:
Page 202 and 203:
FLT3 Mutations in AML 189 12 FLT3 M
Page 204 and 205:
FLT3 Mutations in AML 191 3.1.1. DN
Page 206 and 207:
FLT3 Mutations in AML 193 Table 2 R
Page 208 and 209:
FLT3 Mutations in AML 195 Fig. 2. D
Page 210 and 211:
FLT3 Mutations in AML 197 10. Kiyoi
Page 212 and 213:
WT1 Expression in AML and MDS 199 1
Page 214 and 215:
WT1 Expression in AML and MDS 201 T
Page 216 and 217:
Page 218 and 219:
WT1 Expression in AML and MDS 205 T
Page 220 and 221:
WT1 Expression in AML and MDS 207 F
Page 222 and 223:
Page 224 and 225:
Page 226 and 227:
Classification of AML by DNA-Oligon
Page 228 and 229:
Page 230 and 231:
Page 232 and 233:
Page 234 and 235:
Page 236 and 237:
Page 238 and 239:
Page 240 and 241:
Page 242 and 243:
Page 244 and 245:
Page 246 and 247:
233 Classification of AML by DNA-Ol
Page 248 and 249:
Page 250 and 251:
Page 252 and 253:
Page 254 and 255:
Classification of AML by Monoclonal
Page 256 and 257:
Page 258 and 259:
Page 260 and 261:
247 Classification of AML by Monocl
Page 262 and 263:
Page 264 and 265:
Page 266 and 267:
Detecting JAK2 V617F Mutation in MP
Page 268 and 269:
Page 270 and 271:
Page 272 and 273:
Page 274 and 275:
Page 276 and 277:
Page 278 and 279:
PRV-1 Overexpression in PV and ET 2
Page 280 and 281:
Page 282 and 283:
Page 284 and 285:
Page 286 and 287:
Page 288 and 289:
Chimerism Analysis 275 18 Chimerism
Page 290 and 291:
Chimerism Analysis 277 1.2. Detecti
Page 292 and 293:
Chimerism Analysis 279 3. Extractio
Page 294 and 295:
Chimerism Analysis 281 14. 310 Gene
Page 296 and 297:
Chimerism Analysis 283 12. Upon com
Page 298 and 299:
Chimerism Analysis 285 Fig. 2. Calc
Page 300 and 301:
Chimerism Analysis 287 4. Allow the
Page 302 and 303:
Chimerism Analysis 289 capillary el
Page 304 and 305:
Chimerism Analysis 291 and recipien
Page 306 and 307:
Chimerism Analysis 293 Table 2 Comm
Page 308:
Chimerism Analysis 295 12. Maas, F.
Page 311 and 312:
298 Index management, 128 AML, see
Page 313 and 314:
300 Index and AML, 213, 236, 238, 2
Page 315 and 316:
302 Index chimerism analysis in sex
Page 317 and 318:
304 Index minimal residual disease
Page 319:
306 Index Stem cell transplantation
show all

Myeloid Leukemia

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?