今天我們一起來拜讀華盛頓大學Christina A. Gurnett課題組在Nature Methods發表的工作，如何進行突變蛋白體篩選。
首先我們來一起看看摘要部分：
Large-scale mutagenesis of target DNA sequences allows researchers to comprehensively assess the effects of single-nucleotide changes. Here we demonstrate the construction of a systematic allelic series (SAS) using massively parallel single-nucleotide mutagenesis with reversibly terminated deoxyinosine triphosphates (rtITP). We created a mutational library containing every possible single-nucleotide mutation surrounding the active site of the TEM-1 β-lactamase gene. When combined with high-throughput functional assays, SAS mutational libraries can expedite the functional assessment of genetic variation。
大家讀完摘要，是否可以用一句話進行概括呢？
簡要的說，他們開發了一種新的突變方法，這種方法可以實現包括所有可能的突變。
我們來一個反過來的練習，從這一句話，如果是你，你來寫個摘要。有的小夥伴要說了，這可怎麼寫呀，又不是我做的課題，可是就算是你做的課題，你真的寫的出來？
我們不妨細細的想一想，
［突變方法可以幹嘛］他們開發了一種新的突變方法，這種方法可以實現［用什麼方法實現的］包括所有可能的突變。［用什麼例子來驗證，例子得到什麼好的結果］
好的，我們再考慮一下，如何用英文來說這幾句話。
1.Large-scale mutagenesis of target DNA sequences allows researchers to comprehensively assess the effects of single-nucleotide changes.
2.Here we demonstrate the construction of a systematic allelic series (SAS) using massively parallel single-nucleotide mutagenesis with reversibly terminated deoxyinosine triphosphates (rtITP)
3.We created a mutational library containing every possible single-nucleotide mutationsurrounding the active site of the TEM-1 β-lactamase gene.
4.When combined with high-throughput functional assays, SAS mutational libraries can expedite the functional assessment of genetic variation。
其實就四句話，針對這四句話，想練習SCI論文寫作的同學，不妨再細看看每句話的結構，其實基本都是簡單句。簡單句只要我們抓住主謂賓就可以了。
這裡常見的被使用的動詞：
demonstrate allow assess create expedite
常常積累，做一個有心人，就可以快速的提高：
1.allows researchers to
2.demonstrate the construction of
3.When combined with
讀完了摘要，大家是不是躍躍欲試，希望儘快讀懂文章？
讓我們繼續前進：
Nature Methods 的Brief Communications文章並沒有嚴格的段落劃分，文章的第一段和第二段我們可以視作Introduction部分：
The difficulty of assessing the effects of genetic variants in the human population is a major obstacle confronting precision medicine. The power and utility of rapid, accurate mutation library production has recently been demonstrated1, 2, 3, 4, 5. Several large-scale mutagenesis methods have been used to screen both genes and noncoding loci. Mutagenesis libraries have been created by using a variety of methods, including error-prone or inosine-containing PCR, or by using synthetic oligonucleotides1, 2, 3, 4, 5, 6, 7, 8, 9, 10. However, each of these approaches has its limitations. While error-prone PCR is inexpensive, products often have multiple mutations and high transition:transversion ratios; and the production of oligonucleotides requires substantial up-front financial investment.
大家讀完，再看看紅色的部分，是不是發現有很多固定的語句可以為我們所用？
還是那句話，積累才是硬道理！
The difficulty of ＊＊＊ is a major obstacle confronting＊＊＊
The power and utility of＊＊＊
However，each of these approaches has its limitations
Here we demonstrate the construction of a SAS by performing mutagenesis of segments of DNA using rtITPs. By combining cycle termination used in Sanger sequencing, reversible termination used in Illumina sequencing, and inosine's ability to base pair with each of the four bases, we have systematically incorporated single inosine molecules into DNA molecules, allowing the introduction of one (and only one) mutation per molecule during PCR amplification.
第二段，則是對全文對方法進行了概括，讀到了這裡，想必小夥伴們，已經對這篇文章的方法有了一個初步的認識：
在這篇文章中，科學家們利用在Sanger 測序中使用的循環終止的方法，利用inosine可以與四種鹼基都可以配對，從而在基因組中植入一個inosine，在接下來的PCR擴增過程中，就可以在每個位置進行均勻突變！
具體我們來研究一下細節：
由於adenosine deaminase無法將帶有三個磷酸的ATP進行有效的脫氨基反應，因此科學家們，首先使用鹼性磷酸酶去處磷酸集團，其次，科學家們使用adenosine deaminase脫去氨基，然後再使用磷酸酶生成rtITP
當在PCR中植入rtITP後，因為rtITP無法繼續發生反應。然後去除去除氨基，這樣就可以繼續延伸，這樣就可以保證在每一個PCR的分子中，有且只有一個I鹼基。
科學家在附件中也附上了詳細的protocol哦，快快行動起來吧～～
