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1.发明公开
公开(公告)号：EP0359545A2
公开(公告)日：1990-03-21
申请号：EP89309287.4
申请日：1989-09-13
发明人： Jayaraman, Krishna c/o Eastman Kodak Company , Burdick, Brent Arthur c/o Eastman Kodak Company , Oakes, Fred Terry c/o Eastman Kodak Company
CPC分类号： C12N15/10 , C07K14/80 , C12Q1/6806 , C12Q1/686 , C12Q2565/125 , C12Q2533/107 , C12Q2521/501
摘要： A method of synthesizing double-stranded DNA sequences is disclosed. The method comprises the steps of:
(a) preparing a first series of oligodeoxyribonucleotide fragments which, when joined in proper sequence, form a DNA coding strand;
(b) preparing a second series of oligodeoxyribo- nucleotide fragments which, when joined in proper sequence, form a DNA strand complementary to the coding strand;
(c) compelling hydrogen bonding and ligation in proper sequence between the first and second series of oligodeoxyribonucleotide fragments prepared in steps (a) and (b) in a single reaction to produce the entire double-stranded DNA sequence;
(d) treating the double-stranded DNA sequence with one oligonucleotide primer for each strand under hybridizing conditions;
(e) polymerizing an extension product of each primer that is complementary to each strand of the double-stranded DNA sequence which is a template for forming the primer extension product;
(f) denaturing the product of step (e) to separate the primer extension products from their respective templates to form four separate single-stranded DNA sequences;
(g) treating the denatured product of (f) with oligonucleotide primers, such that a primer extension product is synthesized using each of the single strands produced in step (f) as a template resulting in amplification of the double-stranded DNA sequence; and
(h) repeating steps (d), (e), (f) and (g) until the desired quantity of the double-stranded DNA sequence is formed.摘要翻译： 公开了合成双链DNA序列的方法。 该方法包括以下步骤：（a）制备第一系列寡脱氧核糖核苷酸片段，当以适当顺序连接时形成DNA编码链; （b）制备第二系列的寡脱氧核糖核苷酸片段，当以合适的顺序连接时，形成与编码链互补的DNA链; （c）在单个反应中在步骤（a）和（b）中制备的第一和第二系列寡脱氧核糖核苷酸片段之间以合适的顺序进行引人氢键和连接以产生整个双链DNA序列; （d）在杂交条件下，每条链用一条寡核苷酸引物处理双链DNA序列; （e）使作为形成引物延伸产物的模板的双链DNA序列的每条链互补的每个引物的延伸产物聚合; （f）使步骤（e）的产物变性以将引物延伸产物与其各自的模板分离以形成四个单独的单链DNA序列; （g）用寡核苷酸引物处理（f）的变性产物，使得使用步骤（f）中产生的单链中的每一条合成引物延伸产物作为模板，导致双链DNA序列的扩增; 和（h）重复步骤（d），（e），（f）和（g），直到形成所需量的双链DNA序列。 一世
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公开(公告)号：EP0359545A3
公开(公告)日：1992-01-15
申请号：EP89309287.4
申请日：1989-09-13
发明人： Jayaraman, Krishna c/o Eastman Kodak Company , Burdick, Brent Arthur c/o Eastman Kodak Company , Oakes, Fred Terry c/o Eastman Kodak Company
CPC分类号： C12N15/10 , C07K14/80 , C12Q1/6806 , C12Q1/686 , C12Q2565/125 , C12Q2533/107 , C12Q2521/501
摘要： A method of synthesizing double-stranded DNA sequences is disclosed. The method comprises the steps of:
(a) preparing a first series of oligodeoxyribonucleotide fragments which, when joined in proper sequence, form a DNA coding strand; (b) preparing a second series of oligodeoxyribo- nucleotide fragments which, when joined in proper sequence, form a DNA strand complementary to the coding strand; (c) compelling hydrogen bonding and ligation in proper sequence between the first and second series of oligodeoxyribonucleotide fragments prepared in steps (a) and (b) in a single reaction to produce the entire double-stranded DNA sequence; (d) treating the double-stranded DNA sequence with one oligonucleotide primer for each strand under hybridizing conditions; (e) polymerizing an extension product of each primer that is complementary to each strand of the double-stranded DNA sequence which is a template for forming the primer extension product; (f) denaturing the product of step (e) to separate the primer extension products from their respective templates to form four separate single-stranded DNA sequences; (g) treating the denatured product of (f) with oligonucleotide primers, such that a primer extension product is synthesized using each of the single strands produced in step (f) as a template resulting in amplification of the double-stranded DNA sequence; and (h) repeating steps (d), (e), (f) and (g) until the desired quantity of the double-stranded DNA sequence is formed.
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