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查看更多产品信息 SITE-SATUR-MUT G16 500 BP B - FAQs (838085DE)
20 个常见问题解答
对GeneArt突变服务，我们需要你想进行突变处理的基因的DNA或氨基酸序列，计划使用的宿主信息（如果你在需求中选择包括了基因优化，这一点很重要），你所需要在5 & # 146;/ 3 & # 146;端加上的限制性酶切位点和/或在内部避免的酶切位点信息，以及是否需要添加的任何其它基序(例如，Kozak序列，终止密码子，等等)。
简并文库有三条重要的标准，并且在所有GeneArt简并文库（不包括GeneArt Strings DNA文库）的质量控制中执行：
•非简并部分保持最大序列完整性
•简并位置达到最大序列变异性并达到所要求的核苷酸分布
•文库多样性最大化
•提高或调整启动子强度或特异性
•提高或调节蛋白稳定性
•修改或组合酶的特征
•提高受体、配体或抗体的结合亲和性
•优化或改变信号肽效率
•破坏蛋白功能而同时保留其免疫原性
•组合并选择天然多态性
•提高蛋白半衰期
是的。要达到这一目的，请将您自己的载体或商业化载体寄给我们。
不需要任何材料。为了创建文库，我们所需要的只是在线提交序列文件，以及您想进行随机化的位置信息和位点特征。我们可以通过我们的在线订购系统为您的项目提供报价（https://www.thermofisher.com/order/geneartgenes/projectmgmt）。如果有需要，您可以在项目开始前将有关您文库要求的细节信息提供给我们的合成科学家。
如果您需要一个特殊设计的简并文库，请发送email到geneartsupport@lifetech.com。我们将评估任何项目并且大多数情况下将为您找到一个合适解决方案。
•组合文库（最多1,011个突变体）：多个密码子同时随机化，TRIM技术可选
•位点饱和突变（最多20个突变）：对单一密码子进行各种可能的非野生型随机化突变
•顺次突变文库（密码子数X20个突变）：连续位点饱和突变
•可控随机化（最多1,011个突变）：以固定频率进行无偏向性的随机替换
•所有已知人SH3结构域的cDNA克隆
•截短片段文库：用户定义的截短片段，不含移码突变
•GeneArt Strings DNA文库：可包含最多3个区域的核苷酸随机化的线性DNA片段
有以下原因：
•因为您已经知道某个特定氨基酸替换会破坏您的目的蛋白的功能（例如，互补决定区（CDR）的半胱氨酸）。
•因为一个蛋白可能的突变惊人的多，已经超过了最高通量的筛选能力的几个数量级。无用的突变——例如发生在蛋白非重要区域的突变或引起读码框移位或产生终止密码子的突变——越少，则找到想要的表型的突变的机率越大。
•一些筛选实验是需要成本和较多人力的，因此，筛选较少的克隆可以节省时间和金钱。
传统的创建简并文库的方法（如易错PCR）引入了许多不想要的突变。并且一些方法（像DNA shuffling等一些方法通常不能产生直接相邻突变的组合。然而，合成组合文库可以将突变的引入限制在特定区域，并规定精确的发生频率。此外，相邻突变的重组将不受它们距离远近的影响。
这一问题的答案取决于您的项目细节。通常而言，文库的多样性越低越好，即仅针对基因/蛋白的几个可能在功能上比较重要的区域进行简并设计。下列信息可有助于决定哪些区域比较重要：晶体结构，保守基序，有无同源序列，等等。
For GeneArt mutagenesis services, we will need the DNA or amino acid sequence of the gene you would like to mutagenize, the host organism you plan to use (this is important for gene optimization if you choose to include it with your request), the restriction sites you need at the 5'/3' ends and/or to avoid internally, and whether or not you want any other added motifs (e.g., Kozak sequence, stop codons, etc.).
Three criteria are important for degenerate libraries and are quality-controlled in all GeneArt degenerate libraries (excluding the GeneArt Strings DNA Libraries):
- Maximum sequence integrity of the non-degenerate parts
- Maximum sequence variation of the degenerate positions with the requested nucleotide distribution
- Maximum library diversity
Here are some examples:
- Increase or adjust promoter strength or specificity
- Enhance or modulate protein stability
- Modify or combine enzyme properties
- Increase binding affinities of receptors, ligands, and antibodies
- Optimize or alter signal peptide efficiencies
- Destroy protein function while retaining immunogenicity
- Combine and select natural polymorphisms
- Increase protein half life
- Adjust thermal stability
Yes. For this purpose, please send in your own customized vector or a commercially available vector.
No material is necessary. For library creation, all we need is the sequence file, submitted electronically, and information about the position and nature of the sites you want to randomize. We can provide a quote for your project through our online ordering system (https://www.thermofisher.com/order/geneartgenes/projectmgmt). If needed, you can then relate detailed information about your library request to our production scientists prior to starting the project.
If you require a degenerate library with an unusual design please send an email to geneartsupport@lifetech.com. We will consider any project and in the vast majority of cases will find a solution to fulfill your requirements.
Combinatorial Libraries (up to 1,011 variants): Simultaneous randomization of multiple codons, TRIM technology optional
Site-Saturation Mutagenesis (up to 20 variants): Randomization of a single codon with every possible non–wild type variant
Sequential Permutation Libraries (# of codons x 20 variants): Successive site-saturation mutagenesis
Controlled Randomization (up to 1,011 variants): Unbiased random substitutions with defined frequency
Cloned cDNA of all known human SH3 domains
Truncation Libraries: Customer-defined truncations without out-of-frame mutations.
GeneArt Strings DNA Libraries: linear DNA fragments that can contain up to three regions of randomized nucleotides
There are a number of reasons:
- Because you already know that certain amino acid substitutions disturb the function of your protein (e.g., cysteines in complementarity determining regions (CDRs)).
- Because the number of possible variants of a protein is astronomically high, exceeding the capacity of even the highest-throughput screening capabilities by many orders of magnitude. The fewer useless mutations, such as those occurring in less important regions of the protein or that cause frame shifts or stop codons, the better your chances of finding a variant that results in the desired phenotype.
- Some screening assays are cost and labor intensive; thus, screening fewer clones saves time and money.
Conventional protocols for degenerated library creation (e.g., error-prone PCR) incorporate many unwanted mutations. Moreover, methods like DNA shuffling cannot typically cause recombination of directly adjacent mutations. Synthetic combinatorial libraries, on the other hand, limit the introduction of mutations to defined regions at the precise frequencies requested. In addition, adjacent mutations will be recombined (shuffled) independent of their proximity.
The answer depends on the specifics of your project. In general, it is advantageous to keep the diversity of a library as low as possible, targeting only the regions of a gene/protein that are likely to be functionally important. The following information can help determine this: crystal structure, conserved motifs, presence of homologs, etc.