AmplideX® PCR/CE C9orf72 Kit
The AmplideX PCR/CE C9orf72 Kit (RUO) is a research product for the detection of GGGGCC repeats in the C9orf72 gene. These reagents provide a single-tube PCR approach based on a Repeat-Primed PCR (RP-PCR) design to profile repeat sequences in the C9orf72 gene.
Features & Benefits
Asuragen has established the market-leading technology for the reliable amplification and analysis of CGG triplet repeats in the FMR1 gene. The AmplideX PCR/CE C9orf72 Kit (RUO) is an expansion of this technology into the neurogenetics space. The kit provides clinical and pharmaceutical researchers with a reliable and reproducible method for high-resolution genotyping of hexanucleotide repeat expansions in the C9orf72 gene, which is of increasing interest in frontotemporal dementia (FTD) – the second most common form of early onset dementia after Alzheimer’s Disease – and amyotrophic lateral sclerosis (ALS).
Reduced ComplexityAnalysis of the C9orf72 gene has been simplified through:
- Implementation of proprietary 2-in-1 PCR solution for amplifying GC-rich regions
- A single sourced kit consisting of all PCR reagents needed for C9orf72 repeat amplification
- Streamlined workflow with minimal hands-on time
Optimized WorkflowValuable operator hands-on time has been significantly reduced through:
- Single PCR reaction for both sizing and screening
- Direct injection of PCR products (no PCR clean up) to Capillary Electrophoresis platforms
- Decreased need for Southern blot analysis
Quality PerformancePerforming C9orf72 analysis with greater sensitivity and accuracy:
- Five-fold improvement in length of alleles sized: accurate sizing up to 145 repeats
- Detection of alleles greater than 145 repeats
- Reveal low-level mosaicism and minor alleles
*For Research Use Only. Not for use in Diagnostic procedures.
Analytical Characteristics
Analytical characteristics of AmplideX PCR/CE C9orf72 Kit:
- Accurately sizes up to 145 repeats (Figure 1)
- Detects alleles >145 repeats (Figure 2)
- Resolves zygosity (Figure 3)
Figure 1: Coriell cell line ND06769: The AmplideX Kit correctly sizes a primary allele at 13 repeats and a minor allele at 44 repeats. The expanded allele is also detected in the electropherogram.
Figure 2. Coriell cell line ND06769: Alleles up to 145 repeats can be detected above background and correctly sized, alleles above 145 repeats are amplified and detected as expanded.
Figure 3: Side by side comparison of 2 Coriell cell lines: ND08619 (7,7 homozygous) and ND12102 (7,expanded heterozygous): Extension of the repeat-primed peak pattern beyond the shorter allele clearly differentiates the heterozygous sample from homozygous sample.
Ordering
Product Name | Number of Reactions | Catalog Number |
---|---|---|
AmplideX® PCR/CE C9orf72 Kit (RUO) | 50 | 49581 |
T 1-877-777-1874; 512-681-5200 F 512-681-5202 E orders@asuragen.com
AmplideX® PCR/CE C9orf72 Kit
The AmplideX PCR/CE C9orf72 Kit* is a research product for the detection of GGGGCC repeats in the C9orf72 gene. These reagents provide a single-tube PCR approach based on a Repeat-Primed PCR (RP-PCR) design to profile repeat sequences in the C9orf72 gene.
Features & Benefits
Asuragen has established the market-leading technology for the reliable amplification and analysis of CGG triplet repeats in the FMR1 gene. The AmplideX PCR/CE C9orf72 Kit (RUO) is an expansion of this technology into the neurogenetics space. The kit provides clinical and pharmaceutical researchers with a reliable and reproducible method for high-resolution genotyping of hexanucleotide repeat expansions in the C9orf72 gene, which is of increasing interest in frontotemporal dementia (FTD) – the second most common form of early onset dementia after Alzheimer’s Disease – and amyotrophic lateral sclerosis (ALS).
Reduced ComplexityAnalysis of the C9orf72 gene has been simplified through:
- Implementation of proprietary 2-in-1 PCR solution for amplifying GC-rich regions
- A single sourced kit consisting of all PCR reagents needed for C9orf72 repeat amplification
- Streamlined workflow with minimal hands-on time
Optimized WorkflowValuable operator hands-on time has been significantly reduced through:
- Single PCR reaction for both sizing and screening
- Direct injection of PCR products (no PCR clean up) to Capillary Electrophoresis platforms
- Decreased need for Southern blot analysis
Quality PerformancePerforming C9orf72 analysis with greater sensitivity and accuracy:
- Five-fold improvement in length of alleles sized: accurate sizing up to 145 repeats
- Detection of alleles greater than 145 repeats
- Reveal low-level mosaicism and minor alleles
Analytical Characteristics
Analytical characteristics of AmplideX PCR/CE C9orf72 Kit:
- Accurately sizes up to 145 repeats (Figure 1)
- Detects alleles >145 repeats (Figure 2)
- Resolves zygosity (Figure 3)
Figure 1: Coriell cell line ND06769: The AmplideX Kit correctly sizes a primary allele at 13 repeats and a minor allele at 44 repeats. The expanded allele is also detected in the electropherogram.
Figure 2. Coriell cell line ND06769: Alleles up to 145 repeats can be detected above background and correctly sized, alleles above 145 repeats are amplified and detected as expanded.
Figure 3: Side by side comparison of 2 Coriell cell lines: ND08619 (7,7 homozygous) and ND12102 (7,expanded heterozygous): Extension of the repeat-primed peak pattern beyond the shorter allele clearly differentiates the heterozygous sample from homozygous sample.
Ordering
Product Name | Number of Reactions | Catalog Number |
---|---|---|
AmplideX® PCR/CE C9orf72 Kit (RUO) | 50 | 49581 |
T 1-877-777-1874; 512-681-5200 F 1-512-681-5202 E orders@asuragen.com
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逆转录(reverse transcription)是以RNA为模板合成DNA的过程,即RNA指导下的DNA合成。是RNA病毒的复制形式,需逆转录酶的催化。艾滋病病毒(HIV)就是一种典型的逆转录病毒。
逆转录与反转录严格意义上来说没有什么区别,但是逆转录是RNA类病毒自主行为,在整合到宿主细胞内以RNA为模板形成DNA的过程;反转录是进行基因工程过程中,人为地提取出所需要的目的基因的信使RNA,并以之为模板人工合成DNA的过程。二者虽同为RNA→DNA的过程,但地点不同,相对性的来说,逆转录在体内,反转录在体外。
没有序列识别的功能,他就是简单的催化相邻DNA链的5-P末端和3'-OH末端以磷酸二酯键结合的反应,需ATP作辅酶。 不仅可以催化粘性末端之间或平滑末端之间的DNA的连接,也催化DNA与RNA之间以及少数RNA之间的连接。
反转录酶(Reversetranscripatase)是以RNA为模板指导三磷酸脱氧核苷酸合成互补DNA(cDNA)的酶。哺乳类C型病毒的反转录酶和鼠类B型病毒的反转录酶都是一条多肽链。鸟类RNA病毒的反转录酶则由两上亚基结构。真核生物中也都分离出具有不同结构的反转录酶。
只要是细胞,就得走向衰老,现代研究表明,细胞的衰老和端粒的变短有关系。端粒就是DNA,随着细胞分裂次数的增多,端粒在不断变短,端粒酶就是组织端粒变短的。然而正常细胞中,端粒酶的活性受抑制,端粒酶是怎么染端粒变长的呢?实际上端粒酶是由RNA组成的,可以根据碱基互补配对逆转形成DNA,使得变短的端粒变长。所以说端粒酶可以逆转录形成端粒,使端粒不减短!
RNA聚合酶Ⅰ存在于核仁中,转录rRNA顺序。RNA聚合酶Ⅱ存在于核质中,转录大多数基因,需要“TATA”框。RNA聚合酶Ⅲ存在于核质中,转录很少 RNA聚合酶几种基因如tRNA基因如5SrRNA基因。有些重复顺序如Alu顺序可能也由这种酶转录。
在进行RT反应之前,应考虑以下几个方面:
1、RNA
成功的cDNA合成来自高质量的RNA,高质量的RNA至少应保证全长并且不含逆转录酶的抑制剂,如EDTA或SDS。在提取RNA的过程中,要特别防止RNase的污染,同时在逆转录反应中经常加入RNase抑制剂以增加cDNA合成的长度和产量。RNase抑制剂要在第一链cDNA合成反应中,在缓冲液和还原剂(如DTT)存在的条件下加入,因为cDNA合成前的过程会使抑制剂变性,从而释放结合的可以降解RNA的RNase。蛋白RNase抑制剂仅防止RNaseA,B,C对RNA的降解,并不能防止皮肤上的RNase,因此尽管使用了这些抑制剂,也要小心不要从手指上引入RNase,实验过程中经常更换新手套。
2、引物的选择
OligodT
选择OligodT时,要求RNA必须有PolyA,所以真核生物的mRNA都适用。适合长链甚至全长mRNA的RT,所以对RNA样品的质量要求较高,最好不要有明显的DNA污染、RNA降解和RNA断裂。假如想探索新的mRNA进行RT反应,建议推荐使用OligodT引物。使用OligodT引物要比随机引物和特异性引物的稳定性要好。
随机引物
适合各种RNA的RT,尤其适合模板丰度很低的情况(比如某个gene表达量很低)。选择随机引物时,第一链cDNA合成反应中就是以所有的RNA为模板,然后进行PCR反应时设计引物进行特异性扩增。同时要注意随机引物的量和总RNA量之间的关系,一般建议每5μg总RNA的随机引物的用量为50ng,如果每5μg总RNA的随机引物的用量超过250ng,可能会导致小片段产物(<500bp)的增加和长片断、全长产物产物的降低。
特异性引物
特异性引物只能用你设计引物时的下游引物做RT,引物设计质量影响RT的结果,而且不同引物退火温度本来就不相同,所以按照说明书按照一个温度做不是最佳选择,一般不推荐。向左转|向右转
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