1图位克隆

• A gene can be moved from one organism to another. An organism containing a "foreign" gene via genetic engineering process is called transgenic. • Transgenic organisms can be used either for laboratory research to advance our understanding of biological processes or for specialized industrial applications – for examples:
主要技术环节 遗传作图 近等基因系 BSA 筛选连锁的分子标记 Hale Waihona Puke Baidu 目的基因的精细定位和作图 目的基因分离 目的基因鉴定
Genetic map – in this case, the maps of potato and tomato are aligned, 遗传作图 Genetic Map
图位克隆法步骤
(c ) Chromosome walking and landing: -- Chromosome walking relies on isolation of a DNA fragment at or near an end of a cloned insert for use as a probe to screen the library and identify more clones. -- Chromosomal landing: It is based on the principle that the expected average between-marker distances can be smaller than the average insert length of a clone library containing the gene of interest (d) Gene identification: Genetic complementation through transformation.
第三节、基因克隆策略
一、目的与意义
• Isolation of gene enables the determination of its nucleotide sequence. determining –intron number and position、 promoter elements，etc.
• A comparison of DNA sequences between genes can lead to insights in gene evolution.
• Converting the DNA sequence of a gene into amino acid sequence by using the genetic code leads to a determination of the structure of the protein product of the gene, and from this knowledge, the function of the gene can be inferred.
and the markers align perfectly except for a few inversions (indicated by arrows)
物理图谱（physical map)
物理图谱 基因所在DNA区域的物理特征 限制性酶切图谱
• Development of insect resistant crops • Production of human insulin in transgenic bacteria carrying appropriate human gene
二、常用的克隆策略
• • • • • • • • • 图位克隆法 标签法 同源序列法 功能克隆法 mRNA差异比较 利用EST数据库和更新的SAGE法 基因芯片 RNA-seq 基因文库及目的基因分离
1.图位克隆（Map-based cloning）法（未 知序列的基因克隆）
图位克隆(Map-based cloning)又称定位克隆(positional cloning)，1986年， 剑桥大学的Alan Coulson提出，用该方法分离基因是根据目的基因在染 色体上的位置进行基因克隆的一种方法。 在利用分子标记技术对目的基因进行精确定位的基础上，使用与目的基 因紧密连锁的分子标记筛选DNA文库，从而构建目的基因区域的物理图 谱，再利用此物理图谱通过染色体步行逼近目的基因或通过染色体登陆 的方法最终找到包含该目的基因的克隆，最后通过遗传转化和功能互补 验证最终确定目的基因的碱基序列。
图位克隆方法分离基因的优点是无需预先知道基因的DNA顺序，也无需 预先知道其表达产物的有关信息。
图位克隆法步骤
‫ ٭‬90年代初发展起来的基因克隆技术。很多分离克隆的 基因都是通过这种方法获得的。
‫ ٭‬图位克隆法是基于基因在遗传图谱上的位置而分离克 隆基因的方法。 ‫ ٭‬图位克隆法包括以下步骤： (a) Target gene mapping: a high resolution genetic map with average distance is less than 5 cM. (b) Physical mapping: a map of the locations of identifiable landmarks on DNA, regardless of inheritance. Distance is measured in base pairs other than genetic recombination (in cM).