ACGT A)A sequencing gel with four separate lanes.The sequence begins,reading from the bottom. B)An example of -GCGACAT- sequencing by use of +ddA +ddG +ddC +ddT fluorecent -GCGACA-GCG -GCGAC -GCGACAT -GCGA -G -GC dideoxynucleoside triphosphates..采用荧光 Mix and electrophorese 双脱氧核苷三磷酸 C)Part of an automated DNA 三 (a) (b) sequencing run GCGACATCACTCCAGCTTGAAGCAGTTOTTCTCGTCTTCTGTTTTGTCTAACTTGTCTTCCTTCTTCTCTTCCTGTTTAAGAAGAGAA 500 510 520 530 540 550 560 570 580
A) A sequencing gel with four separate lanes. The sequence begins, reading from the bottom. B) An example of sequencing by use of fluorecent dideoxynucleoside triphosphates. 采用荧光 双脱氧核苷三磷酸 C) Part of an automated DNA sequencing run
Shotgun sequencing鸟枪法测序 Need random fragments The fragments are sequenced without knowing the order or orientation of the cloned DNA.or even if one has already sequenced this region. Generally 7-to 10-fold coverage > The sequences obtained will then be analyzed by computer,the computer will seek overlapping sequences to construct a complete sequence of the entire genome. Chen Feng,Lecture of Microbiology
Chen Feng, Lecture of Microbiology Need random fragments The fragments are sequenced without knowing the order or orientation of the cloned DNA, or even if one has already sequenced this region. Generally 7- to 10-fold coverage The sequences obtained will then be analyzed by computer, the computer will seek overlapping sequences to construct a complete sequence of the entire genome. Shotgun sequencing 鸟枪法测序
H.influenzae chromosome Whole-genome shotgun Sonication sequencing DNA fragments Agarose gel electrophoresis This approach may be broken into four stages: of fragments and DNA size markers I.Library construction:large chromosomes were randomly broken into fairly small fragments Fragment purification using ultrasonic waves;these fragments were from gel attached to plasmid vectors.文库构建 DNA fragments (about 2kb) ‖.Random sequencing:随机测序 Clonal library preparation Ill.Fragment alignment and gap closure:the sequenced DNA fragments were clustered and Sequence the clonal assembled into longer stretches of sequence by inserts,particularly the end sequences. comparing sequence overlaps between End sequences fragments..片段排列 Construct sequence contigs and align using overlaps;fill IV.Editing:the sequence was carefully proofread. in gaps 辑 3 Chen Feng,Lecture of Microbiology
Chen Feng, Lecture of Microbiology Whole-genome shotgun sequencing This approach may be broken into four stages: I. Library construction: large chromosomes were randomly broken into fairly small fragments using ultrasonic waves; these fragments were attached to plasmid vectors. 文库构建 II. Random sequencing: 随机测序 III. Fragment alignment and gap closure: the sequenced DNA fragments were clustered and assembled into longer stretches of sequence by comparing sequence overlaps between fragments. 片段排列 IV. Editing: the sequence was carefully proofread.编 辑
How to get random fragments? Restriction enzyme:restriction sites are not random,but by cutting incompletely with an enzyme that recognizes a short sequence,one can approach random digestion. 制性酶不完全剪切获得随机片段 Mechanically sheared random fragments: phosphodiester bonds of long DNA molecules in solution could be broken by shearing stresses during the process of purification.机械剪切获得随机片段 ⑧ Broken into small fragments using ultrasonic waves Chen Feng,Lecture of Microbiology
Chen Feng, Lecture of Microbiology How to get random fragments? Restriction enzyme: restriction sites are not random, but by cutting incompletely with an enzyme that recognizes a short sequence, one can approach random digestion.限 制性酶不完全剪切获得随机片段 Mechanically sheared random fragments: phosphodiester bonds of long DNA molecules in solution could be broken by shearing stresses during the process of purification.机械剪切获得随机片段 Broken into small fragments using ultrasonic waves
454 sequencing system Recent technological advance Generates data 100x faster than Sanger method 454 relies on two major advances Massively parallel liguid handling and pyrosequencing Light is released each time a base is added to DNA strand Instrument actually measures release of light Can only handle short stretches of DNA Chen Feng,Lecture of Microbiology
Chen Feng, Lecture of Microbiology 454 sequencing system Recent technological advance Generates data 100 faster than Sanger method 454 relies on two major advances • Massively parallel liquid handling and pyrosequencing • Light is released each time a base is added to DNA strand • Instrument actually measures release of light • Can only handle short stretches of DNA