焦磷酸测序技术 依次加入不同的 Iterative additions dNTP dATP一dCTP→dGTP→dTP pyrosequencing dCTP 焦磷酸测序技术是 无反应dNTP被降解 种新的实时DNA测序 AGCGTCATGGTTAAATTG. 技术。它在DNA聚合 TCGCAGTAC NMP 酶、三磷酸腺苷硫酸 dNTP dNDP 引物 PPi 化酶、荧光素酶和三 三磷酸腺苷硫酸化酶 磷酸腺苷双磷酸酶 ATP Sulfurylase 三磷酸腺苷双磷酸酶 4种酶的协同作用 ,+403→AP+8 下,使引物延伸聚合 D-luciferin uciferase luciferin. AMP +O 脱氧核糖核酸.dNTP 荧光素 Luciferase 荧光素酶 释放焦磷酸盐PPi Nucleotide sequence Luciferase+ oxyluciferin+ AMP+ cO 2 PPi转换为三磷酸腺 荧光强度可以判别是否连续 苷(ATP)、ATP产 相同的碱基序列 生荧光信号与dNTP HPV18 和ATP的降解等化学 反应偶联起来,检测 CCD camera or 结果准确可靠。 Nucleotide added
焦磷酸测序技术 pyrosequencing 焦磷酸测序技术是一 种新的实时DNA 测序 技术。它在DNA 聚合 酶、三磷酸腺苷硫酸 化酶、荧光素酶和三 磷酸腺苷双磷酸酶 4 种酶的协同作用 下，使引物延伸聚合 脱氧核糖核酸. dNTP 释放焦磷酸盐PPi、 PPi转换为三磷酸腺 苷（ATP）、ATP 产 生荧光信号与dNTP 和ATP 的降解等化学 反应偶联起来，检测 结果准确可靠
循环阵列测序-滚环扩增建库 cyclic-array sequencing a In vitro mate-paired library construction universal 滚环复制 sequences 测序文库构建 1 kb genomic 拷贝扩增 DNA fragment Universal linker paired genomic tags Mme酶切为点顺序 NNASSVESNNNNNNNNNNNNNNNNNNNNNN Mmel digestion half mate-paired genomic sequences i: Sheared, size-selected genomic fragments (yellow)are circularized with a linker(red ) bearing Mme I recognition sites. Subsequent steps, which include a rolling circle amplification, yield the 134-to 136-bp mate-paired library molecules
循环阵列测序--滚环扩增建库 cyclic-array sequencing 测 序 文 库 构 建 滚环复制 拷贝扩增
Polymerase colonv. or polony technologies 循环阵列测序PcR建库 I perform multiplex amplification whil maintaining spatial clustering of identical amplicons In emulsion PCr(ePCr),a Emulsion PCR waterin-oil emulsion permits millions of noninteracting amplifications within a millHiliter-scale volume. Amplification products of individual compartments are captured via inclusion of 1-mm paramagnetic beads bearing one of the PCR primers. Any single bead bears thousands of single-stranded copies of the same PCr product, whereas different beads bear the products of different compartmentalized PCr reactions. The beads generated by ePcr have highly desirable characteristics 乳液PR:每一粒乳液含一个磁珠, high signal density, geometric 含一个待扩增的DNA片段 uniformity, strong feature separation, 注: ePCR yields clonal template amplification on and a size that is small but still 1-mm beads resolvable by inexpensive optics
循环阵列测序-PCR建库 Polymerase colony, or polony,technologies perform multiplex amplification while maintaining spatial clustering of identical amplicons. In emulsion PCR (ePCR), a waterin-oil emulsion permits millions of noninteracting amplifications within a milliliter-scale volume . Amplification products of individual compartments are captured via inclusion of 1-mm paramagnetic beads bearing one of the PCR primers . Any single bead bears thousands of single-stranded copies of the same PCR product, whereas different beads bear the products of different compartmentalized PCR reactions . The beads generated by ePCR have highly desirable characteristics: high signal density, geometric uniformity, strong feature separation, and a size that is small but still resolvable by inexpensive optics
循环 C Enrichment& Monolayering专-性分子杂交富集含有扩 放DNA的磁珠,离心收集。 0 阵列 淘汰0000 测序0080980 1.5 cm 富集00*兼兼。(共 与 富集、0"0 测序 芯片 I: Hybridization to nonmagnetic, low-density"capture beads (dark blue) permits enrichment of the amplified fraction(red)of 制作 magnetic ePCR beads by centrifugation. Beads are immobilized and mounted in a flowcell for automated sequencing
循环 阵列 测序 - 富集 与 测序 芯片 制作
循环阵列测序焦磷酸测序 Step 1: load beads with DNA ●o0 Step 2: inject dNTPs, detect light signal Polymerase ATP
循环阵列测序—焦磷酸测序