New-gene manipulication class -xs 2023-课件1（周一）

1. 第五章 基因操作与基因组分析
Gene Manipulation & Genomic Analysis
2023. 05
1

2. 1992-1996 北京师范大学 生物系 学士
1996-1999 北京师范大学 生物系 硕士
1999-2006 美国罗彻斯特大学，细胞、分子和发育生物学 博士
2006-2012 加州大学圣地亚哥分校，博士后
2012.12-至今 中国科学技术大学，生命学院，教授
宋晓元, Ph.D.
2

3. 1. 重组DNA的原理、方法及鉴定 （宋老师讲课）
2. 重组DNA技术的应用及基因组分析 （宋老师讲课）
3. 分组讨论各类基因操作和基因组分析的技术（同学讲课）
3
课程安排

4. 分组讨论
1. 克隆筛选方法及发展
2. 鉴定和分析已克隆的DNA的方法及发展
3. DNA测序技术的进展及应用
4. 遗传图谱绘制技术的发展和应用
5. PCR技术的应用
6. 基因表达和功能的研究技术和方法
7. 基因编辑技术的进展与应用
8. 三维基因组的研究技术发展和应用
9. 非编码RNA研究方法的进展与应用
10. DNA-蛋白质相互作用的技术进展及应用
11. 细胞治疗的进展和应用
12. 基因治疗的进展和应用
按签到表顺序每4人一组（第六组5人），每组选两个方向，
2人找资料，1人做PPT，1人讲PPT （标注好全组成员姓名，
列出各人贡献）。两个方向各人的工作互换。
5 月 28 日 晚 12 点 之 前 将 讲 PPT 发 给 助 教 王 玉 珠 ：
562816066@qq.com；抄送给我：songxy5@163.com
4

5. 重组DNA 技术
5

6. 重组DNA技术
Once the structure of DNA and the genetic code were
unraveled, it became clear that many deep biological
secrets were locked up in the sequence of bases in DNA.
An avalanche of technical discoveries in the 1970s
drastically changed this perspective and has led to
astounding advances in molecular cell biology in the past
several decades based on the analysis and manipulation of
macromolecules, particularly DNA.
6

7. Cloning is to make
many copies of a
segment of DNA such
as a gene.
DNA is cloned by
inserting a DNA
fragment into a
cloning vector to make
a recombinant DNA
molecule (ex vivo)。
7

8. DNA克隆技术中所需要的两个关键酶
Restriction Endonuclease DNA ligase
8

9. Restriction-recognition sites: short DNA sequences
recognized and cleaved by various restriction endonucleases
9

10. Restriction-recognition sites: short DNA sequences
recognized and cleaved by various restriction endonucleases
10

11. 限制性内切酶的分类
EcoR I digestion produces “sticky” ends (粘性末端）
SmaI restriction enzyme cleavage produces "blunt" ends
（平端）
EcoP15I cleaves sites far from the
recognition site
11

12. 互补粘性末端的连接
12

13. 相容性的末端
1. Cleavage with two restriction endonucleases that
produce compatible overhangs.
2. Cleavage with two restriction endonucleases that
produce blunt ends.
3. Cleavage followed by fill-in of overhangs to generate
blunt ends.
13

14. BamHI BglII
5’ GGATCT
3’ CCTAGA
AGATCC3’
TCTAGG5’
相容性的粘性末端
14

15. Sma I EcoR V
CCCATC
GGGTAG
GATGGG
CTACCC
平端
15

16. Creating blunt ends from sticky cut by
T4 DNA polymerase fill-in
GGATCC
CCTAGG
G
CCTAG
GATCC
G
BamHI
GGATC
CCTAG
GATCC
CTAGG
T4 DNA
polymerase
Open question: Other methods?
16

17. 限制性酶切  Produce a 3’-OH and a 5’-Pi
17

18. T4 DNA连接酶 （ligase)
 The combining of 3’-
and 5’- ends requires
at least one of them is
phosphorelated.
18

19. Reducing self ligation by removing the Pi
from the vector by phosphotase
GGATCC
CCTAGG
G
CCTAG
GATCC
G
BamHI
Pi
Pi
G
CCTAG
GATCC
G
OH
OH
OH
OH
OH
OH
Alkaline Phosphatase,
Calf Intestinal (CIP)
19

20. Chemical synthesized oligoes should be
phosphoryated by T4 polynucleotide kinase
to make it clonable
GATCC
G
OH
OH
T4 PNK
GATCC
G
Pi
OH
20

21. Serial DNA cloning can be used to splice
together a set of DNA fragments derived from
different genes.
21

22. The future
Synthetic genomics combines methods for the
chemical synthesis of DNA with computational
techniques to design it. These methods allow scientists
and engineers to construct genetic material that
would be impossible or impractical to produce
using more conventional biotechnological
approaches. For example, using synthetic genomics
it is possible to design and assemble chromosomes,
genes and gene pathways, and even whole genomes.
J. Craig Venter Institute
22

23. J. Craig Venter institute 一个大型的、多学科的、以基因组为重点的组织
The J. Craig Venter Institute was formed in October 2006 through the
merger of several affiliated and legacy organizations — The Institute for
Genomic Research (TIGR) and The Center for the Advancement of
Genomics (TCAG), The J. Craig Venter Science Foundation, The Joint
Technology Center, and the Institute for Biological Energy Alternatives
(IBEA). Today all these organizations have become one large
multidisciplinary genomic-focused organization. With more than 400
scientists and staff, more than 250,000 square feet of laboratory space,
and locations in Rockville, Maryland and San Diego, California, the new
JCVI is a world leader in genomic research.
23

24. Chemical
synthesis of
oligonucleotides
by sequential
addition of
reactive
nucleotide
derivatives in the
3 → 5 direction
24

25. ClonExpress技术
是一种简单、快速并且高效的DNA无缝克隆技术，可将插入片段定向克
隆至任意载体的任意位点。使用任意方式将载体进行线性化，在插入片
段正/反向PCR引物5'端引入线性化载体的末端序列，使得PCR产物5'和
3'最末端分别带有和线性化载体两末端一致的序列(15 - 20 bp)。
这种两端带有载体末端序列的PCR产物和线性化载体按一定比例混合后
，在重组酶的催化下，37℃反应30 min即可进行转化，完成定向克隆
Vazyme
25
多片段拼接无缝克隆技术（by 重组酶）

26. ClonExpress技术
是一种简单、快速并且高效的DNA无缝克隆技术，可将插入片段定向克
隆至任意载体的任意位点。使用任意方式将载体进行线性化，在插入片
段正/反向PCR引物5'端引入线性化载体的末端序列，使得PCR产物5'和
3'最末端分别带有和线性化载体两末端一致的序列(15 - 20 bp)。
这种两端带有载体末端序列的PCR产物和线性化载体按一定比例混合后
，在重组酶的催化下，37℃反应30 min即可进行转化，完成定向克隆
Vazyme
26
Vazyme
多片段拼接无缝克隆技术（by 重组酶）

27. 克隆的筛选
27

28. How to select the correct clone
with the insert?
1. Miniprep + RE digestion
2. Blue-white
3. PCR screening
Final step: Confirm by DNA
sequencing
克隆的筛选
28

29. Blue-White selection
29

30. A cloning vector
 Need an replication ori
 A drug resistance gene for screening
 A polylinker for DNA insertion
 polylinker inside lacZ+ gene to detect
insertion (cause reading frame change) by
color (blue for lacZ+ and white for
lacZ- )
30

31. Plasmid DNA replication
31

32. Plasmid vectors
containing a
polylinker, or
multiple-cloning-
site sequence,
commonly are
used to produce
recombinant
plasmids
carrying
exogenous DNA
fragments.
32

33. Fragments produced by cleavage of the ≈36kb
DNA genome from adenovirus 2 (Ad2) by
EcoRI and another restriction enzyme, HindIII
33

34. Isolation of DNA fragments from a mixture
by cloning in a plasmid vector
Clean up contamination?
34

35. Use  DNA as a vector
 An  head can pack ~37-52 kb of DNA
 Can carry as much as 15 kb foreign DNA
35

36.  bacteriophage undergoes either lytic replication
or lysogeny following infection of E. coli.
36

37. Assembly of
bacteriophage 
virions
Concatemer
（噬菌体）串联体 A concatemer is a
long continuous DNA molecule that
contains multiple copies of the same
DNA sequence linked in series.
37

38. Vector Type Cloned DNA
(kb)
Plasmid 20
 phage 25
Cosmid 45
P1 phage 100
BAC (bacterial artificial
chromosome)
300
YAC (yeast artificial chromosome) 1000
Approximate Maximum Length of
DNA That Can Be Cloned in Vectors
39

39. General procedure for
cloning DNA fragments
in cosmid vectors
40

40. YAC--yeast artificial chromosome
 Eukaryotic chromosome replication requires: two
telomeres, centromere and the origin of replication
 YAC contains: telemeres (TEL), centromere (CEN),
selectable markers (TRP1 and URA3), origin of
replication (ARS), polylinker.
 YAC can carry DNA fragments of hundreds of kp, used
for creating genomic mapping (physical)
41

41. Shuttle vectors
Shuttle vectors are
those that can replicate
in more than one
organisms
An E.coli/yeast shuttle vector
Clone construction use E.coli,
later use shuttle vector to
introduce cloned gene into
Eukaryotic cells
42

42. Genomic Library
43

43. What is a Genomic Library
A collection of clones that contains (at
least) one copy of every DNA sequence in a
genome
How to construct a genomic library
1. Complete/partial digestion of genomic DNA
2. Clone each fragment into a vector
3. Transform host organism
Considerations: the density of digestion sites.
Sequences that may affect vector replicating
44

44. Production of overlapping restriction
fragments by partial digestion of human
genomic DNA with Sau3A
45

45. Construction of a
genomic library of
human DNA in a
bacteriophage 
vector
46

46. chromosome library
Individual chromosomes can be separated by morphologies and sizes.
Once the chromosomes have been sorted and collected, a library of
each chromosome type can be made.
The dyes usually selected are Hoechst
33258, which shows a preference for AT-
rich regions, and chromomycin A3, which
binds to GC-rich regions. A cytometer with
two high-powered argon-ion lasers is
required; one laser is tuned to give UV to
excite the Hoechst dye (blue fluorescence)
and the other to 458 nm to excite the
chromomycin A3 (green fluorescence).
Hoechst 33258为非嵌人性荧光染料,在活细胞中DNA聚AT序列富集区域的小沟处与
DNA结合。活细胞或固定细胞均可从低浓度溶液中摄取该染料。从而使细胞核着色。
故又把此类染料称为DNA探针。Hoechsr-DNA的激发和发射波长分别460nm和550nm
。在荧光显微镜紫外光激发时，Hoechst-DNA发出亮蓝色荧光。需要一个带有两个高
功率氩离子激光器的细胞仪 47

47. Flow cytometry
48

48. PFGE: Pulsed field gel electrophoresis
脉冲场凝胶电泳
在普通的凝胶电泳中，大的DNA分子（>10kb）移动速度接近，很难分离形成
足以区分的条带。在脉冲场凝胶电泳中，电场不断在两种方向（有一定夹角，
而不是相反的两个方向）变动。DNA分子带有负电荷，会朝正极移动。相对较
小的分子在电场转换后可以较快转变移动方向，而较大的分子在凝胶中转向较
为困难。因此小分子向前移动的速度比大分子快。脉冲场凝胶电泳可以用来分
离大小从10kb到10Mb的DNA分子。
49

49. Synthesis of cDNA library
What is cDNA: complementary
DNA made from RNA
cDNAgene: promoter region+exons+
introns
Procedure (mRNA cDNA as an example)
1. The enriching of mRNA
2. Oligo dT as primer
3. Reverse transcriptase--cDNA
4. mRNA degradation
5. Making dsDNA
6. Clone into vector
50

50. Isolation of eukaryotic mRNA
by oligo-dT column affinity
chromatography
51

51. Preparation of a
bacteriophage  cDNA
library
52

52. Why use N(dT) as a primer?
How to clone an intact RNA?
Why not use anti-m7G CAP IP to purify mRNA?
53

53. Primer Extension
 Primer extension works to determine exactly
the 5’-end of a transcript to single-nucleotide
accuracy
 Specificity of this method is due to
complementarity between primer and transcript
 S1 mapping will give similar results but has
limits:
 S1 will “nibble” ends of RNA-DNA hybrid
 Also can “nibble” A-T rich regions that have melted
 Might not completely digest single-stranded regions
(S1 nuclease: digest ss DNA or RNA, not works on dsDNA,
dsRNA, DNA-RNA)
5-54

54. Primer Extension Schematic
 Start with in vivo transcription,
harvest cellular RNA
containing desired transcript
 Hybridize labeled
oligonucleotide [18nt] (primer)
 Reverse transcriptase extends
the primer to the 5’-end of
transcript
 Denature the RNA-DNA
hybrid and run the mix on a
high-resolution DNA gel
 Can estimate transcript
concentration also
5-55

55. Subtractive hybridization
What is the current technique?
56

56. Removing rRNAs
Ribo-Zero kit (Epicenter)
Ribominus kit (Invitrogen)
remove ribosomal RNA (rRNA) using a hybridization/bead
capture procedure that selectively binds rRNA species
using biotinylated capture probes. The probe: rRNA hybrid
is then captured by magnetic beads and removed using a
magnet, leaving the desired rRNA-depleted RNA in solution.
Gel purification
57

57. Small RNA library
by the
ExactSTART™ Small
RNA Cloning Kit
58

58. How to clone a RNA that has PiPiPi at 5’-ends?
(1) Change 3 to 1
(2) Reverse transcription first
59

59. Identifying, Analyzing, and Sequencing Cloned DNA

60. Membrane-hybridization
assay for detecting
nucleic acids
Dot plot

62. Identification of a
specific clone from a 
phage library by
membrane hybridization
to a radiolabeled probe

63. Use of  expression cloning to identify a cloned DNA based
on binding of the encoded protein to a specific antibody

64. A simple E. coli expression vector
utilizing the lac promoter

65. Two-step expression vector system based on
bacteriophage T7 RNA polymerase and T7 late
promoter.
Toxic proteins

66. Separation of DNA fragments of
different lengths by gel
electrophoresis

67. The Southern blot
A technique for detecting the presence of specific DNA sequences following gel
electrophoresis of a complex mixture of restriction fragments.
Southern印迹由Edwin Southern于1975年推出，
是一种检测DNA样品中特定DNA序列的方法

68. Radiolabeling of an oligonucleotide at the 5’ and with phosphorus-32
Radiolabeling of an DNA probe by random priming

69. Mapping the multiple copies
of the recognition site for a
given restriction enzyme in a
DNA fragment.

70. Mapping the recognition sites for two restriction
enzymes relative to each other in a DNA fragment
containing one copy of each site.

71. Construction of a
restriction map
 Mapping restriction
sites of different enzyme
onto a DNA molecule

72. Northern Blot to detect RNA in a mixture

73. Nuclease-protection method for quantitating
specific RNAs in a mixture and mapping them

74. Two methods for mapping the start site for
transcription of a particular gene in a region of
DNA of known sequence.

75. The use of nucleic acid hybridization to determine the region
of a cloned DNA fragment that is present in a mRNA molecule

76. DNA sequencing

77. Maxam-Gilbert sequencing (chemical cleavage method
using double-stranded (ds) DNA)
1. Double-stranded DNA to be sequenced is labelled by attaching a
radioactive phosphorus (32P) group to the 5' end.
2. Using dimethyl sulphoxide and heating to 90oC, the two strands of the DNA
are separated and purified
3. Single-stranded sample is split into separate samples and each is treated
with one of the cleavage reagents.
4. If reactions have been arranged to give only one, or a few, cleavages per
DNA molecule, a nested set of end-labelled DNA fragments of different
lengths is produced.
5. The samples are run together on a sequencing gel which separates the
fragments by electrophoresis depending on their size. DNA bands in the gel
are visualized by autoradiography
6. The DNA sequence is read directly from the gel

78. The Maxam-Gilbert method of
nucleotide sequence
determination is based on
preferential, base-specific
methylation followed by chemical
cleavage to generate a nested
set of end- labeled derivatives.

79. Base specificity
Chemical used for
base alteration
Chemical used for
altered base
removal
Chemical used for
strand cleavage
G Dimethylsulphate Piperidine Piperidine
A+G Acid Acid Piperidine
C+T Hydrazine Piperidine Piperidine
C Hydrazine + alkali Piperidine Piperidine
A>C Alkali Piperidine Piperidine

81. Sanger-Coulson sequencing (chain termination method
using single-stranded (ss) DNA)
1. Sample DNA to be sequenced is cloned into M13 vector DNA to generate ssDNA.
2. A short oligonucleotide primer (usually chemically synthesized and sometimes
labelled ) is added to the ss recombinant DNA.
3. DNA polymerase is then added in the presence of: 4 normal nucleotides: d-ATP, d-
CTP, d-GTP and d-TTP and a low concentration of 4 analogues of the normal
nucleotides (ddNTP, one of which are labelled with 32P) in separate incubation mixes.
4. Complementary strand synthesis occurs away from the primer.
5. Each of the 4 mixes is run together on a sequencing gel which separates the
fragments by electrophoresis depending on their size. DNA bands in the gel are
visualized by autoradiography.
6. The DNA sequence is read directly from the gel in a similar way to a Maxam-Gilbert
sequencing gel.

82. 英国生物化学家Frederick Sanger (2013年11月19日去世，终年95岁)。
Sanger是两届诺贝尔奖的得主，第一次是在1958年因确定胰岛素结构获
得诺贝尔化学奖，第二次是在1980年，因为发明著名的Sanger测序法而
获奖，因此也被称为“基因组学之父”。

83. Structures of ribonucleoside triphosphate (NTP),
deoxyribonucleoside triphosphate (dNTP), and
dideoxyribonucleoside triphosphate (ddNTP)

84. Sanger (dideoxy) method for sequencing DNA fragments

86. AUTOMATED DNA SEQUENCING
It is based on the Sanger-Coulson chain termination method but the 4 different
dideoxy nucleotides (ddA, ddC, ddG and ddT) are fluorescently labelled.
4 different fluorophores are used, all 4 reactions can be run in the same tube.

87. Genome Sequencing

88. Chromosome Walking

89. Genome shotgun sequencing
Strand Sequence
Original AGCATGCTGCAGTCATGCTTAGGCTA
First shotgun sequence
AGCATGCTGCAGTCATGCT-------
-------------------TAGGCTA
Second shotgun sequence
AGCATG--------------------
------CTGCAGTCATGCTTAGGCTA
Reconstruction AGCATGCTGCAGTCATGCTTAGGCTA

90. HUMAN GENOME PROJECT (HGP) 1990
Hierarchical Shotgun
(“map-based”, “BAC-based”,
“clone-by-clone”)
CELERA GENOMICS
1998
Whole Genome Shotgun

91. 人类基因组计划与曼哈顿原子弹计划和阿波罗计划并称为三大科学计划
是由美国科学家于1985年率先提出，于1990年正式启动的。美国、英国、法国、德国、日本
和我国科学家共同参与了这一预算达30亿美元的人类基因组计划。按照这个计划的设想，在
2005年，要把人体内约2.5万个基因的密码全部解开，同时绘制出人类基因的图谱。
2001年2月12日，科学家首次公布“人类基因组图谱”（人类基因组机会成功的里程碑）。
到2003年4月15日，国际人类基因组组织正式宣布，人类基因组计划全部完成。
人类基因组计划”在研究人类过程中建立起来的策略、思想与技术，构成了生命科学领域新
的学科——基因组学，可以用于研究微生物、植物及其他动物。
。。。。。。
功能基因组学

93. metagenomics is the
sequencing and analysis of
DNA of microorganisms
recovered from an environment,
without the need for culturing
them.

94. 可同时对数百万个短序列读长进行测序，
从大规模平行信号测序(MPSS, Brenner
et al.2000)，到454焦磷酸测序法
( Margulies et al.2005)，illumina边合成
边测序（sequencing by synthesis，
SBS），SOLiD连接法测序，Ion Torrent
利用离子敏感场效应晶体管检测pH值变
化的合成测序法，华大智造联合探针锚
定合成测序法（combinatorial probe-
anchor synthesis，cPAS）
1. 454 pyrosequencing
2. Illumina (Solexa) sequencing
3. SOLiD sequencing
4. ION Torrent
Next Generation Sequencing
HUMAN GENOME PROJECT 1990

95. 454 Sequencing, based on sequencing-by-synthesis
Nucleotides are flowed sequentially in a fixed order across the
PicoTiterPlate device during a sequencing run.
During the nucleotide flow, hundreds of thousands of beads each carrying
millions of copies of a unique single-stranded DNA molecule are sequenced
in parallel.
If a nucleotide complementary to the template strand is flowed into a well,
the polymerase extends the existing DNA strand by adding nucelotide(s).
Addition of one (or more) nucleotide(s) results in a reaction that generates a
light signal that is recorded by the CCD camera in the instrument.
The signal strength is proportional to the number of nucleotides incorporated
in a single nucelotide flow.

96. High-throughput droplet PCR

97. Addition of one of the four dNTPs, DNA
polymerase incorporates the correct,
complementary dNTPs onto the template.
This incorporation releases
pyrophosphate (PPi) stoichiometrically.
ATP sulfurylase quantitatively converts
PPi to ATP in the presence of adenosine
5´ phosphosulfate.
This ATP acts as fuel to the luciferase-
mediated conversion of luciferin to
oxyluciferin that generates visible light in
amounts that are proportional to the
amount of ATP. The light produced in the
luciferase-catalyzed reaction is detected
by a camera.
Unincorporated nucleotides and ATP are
degraded by the apyrase, and the
reaction can restart with another
nucleotide.
ssDNA template is hybridized to a sequencing primer and incubated with the
enzymes DNA polymerase, ATP sulfurylase, luciferase and apyrase, and with
the substrates adenosine 5´ phosphosulfate (APS) and luciferin.

98. 454 Sequencing, based on sequencing-by-synthesis

99. Illumina

100. Illumina

103. movie

104. Ion Torrent

105. Ion Torrent

106. Ion Torrent
If a nucleotide matches,
a H+ is released.
If a nucleotide does not
match, no H+ released.
If two bases are incorporated,
two H+ are released.

107. Who invented Ion Torrent?

108. Third Generation Sequencing
DNA sequencing methods currently under development include
labeling the DNA polymerase, reading the sequence as a DNA
strand transits through nanopores, and microscopy-based
techniques, such as AFM or transmission electron microscopy
that are used to identify the positions of individual nucleotides
within long DNA fragments (>5,000 bp) by nucleotide labeling
with heavier elements (e.g., halogens) for visual detection and
recording. (No amplification)

109. 第一代测序技术
Sanger法是基于DNA合成反应的测序技术，又称为SBS法、末端终止法。
1975年由Sanger提出，并于1977发表第一个完整的生物体基因组序列。
核心原理：由于双脱氧核苷酸(ddNTP)的3’位置脱氧，其在DNA的合成过程中
不能形成磷酸二酯键，因此可以用来中断DNA合成反应，在4个DNA合成反应
体系中分别加入一定比例带有放射性同位素标记的ddNTP（分为：
ddATP,ddCTP,ddGTP和ddTTP），通过凝胶电泳和放射自显影，根据电泳带
的位置确定待测分子的DNA序列。
在每个反应体系中，ddNTP相对于dNTP是很少的，所以只有部分新链在不同
的位置特异性终止，最终就会得到一系列长度不一的序列。
作者：生信空间
链接：https://www.jianshu.com/p/c9ade91acced
来源：简书
著作权归作者所有。商业转载请联系作者获得授权，非商业转载请注明出处。

111. 第二代测序技术
以Illumina平台为代表的第二代测序技术实现了高通量测序，有了革命性进展
，使得大规模并行测序成为现实，极大推动了生命科学领域基因组学的发展。
Illumina循环SBS法(cycle SBS)即SBRT(Sequencing By Reversible
Termination,可逆终止)的核心技术是DNA合成的可逆性末端循环，即3'-OH可
逆性的修饰和去修饰。
基本原理：将dNTP的3'-OH以叠氮集团RTG(Reversible Terminating Group,可
逆末端基团)进行修饰；将4种碱基分别与不同的荧光分子连接；DNA合成时，
RTG能起到类似于ddNTP的作用终止反应；每次合成反应终止并读取信号之
后，洗脱RTG和荧光分子，进行下一轮循环。
作者：生信空间
链接：https://www.jianshu.com/p/c9ade91acced
来源：简书

112. 主要过程：
a, DNA待测文库构建
利用超声波把待测的DNA样本打断成小片段，并在这些小片段的两端添加上不
同的接头，构建出单链DNA文库。这些文库中的DNA在通过flowcell(吸附流动
DNA片段的槽道)时会随机附着在flowcell表面的channel上。每个Flowcell有8
个channel，每个channel的表面都附有很多接头，这些接头能和建库过程中加
在DNA片段两端的接头相互配对，并能支持DNA在其表面进行桥式PCR的扩
增。
b,c 桥式PCR以Flowcell表面所固定的接头为模板，进行桥形扩增。经过不断
的扩增和变性循环，最终每个DNA片段都将在各自的位置上集中成束，每一个
束都含有单个DNA模板的很多份拷贝，进行这一过程的目的在于实现将碱基信
号强度放大，以达到测序所需的信号要求。
b,c 桥式PCR以Flowcell表面所固定的接头为模板，进行桥形扩增。经过不断
的扩增和变性循环，最终每个DNA片段都将在各自的位置上集中成束，每一个
束都含有单个DNA模板的很多份拷贝，进行这一过程的目的在于实现将碱基信
号强度放大，以达到测序所需的信号要求。
作者：生信空间
链接：https://www.jianshu.com/p/c9ade91acced
来源：简书

114. 第三代测序技术
以Pacbio平台为代表的SMRT(Single-Molecule Real Time Sequencing,单分子
实时测序)测序技术具有高通量、长读长的特点。
基本原理：Pacbio仍然采用边合成边测序的原理，但实现了两个重要的技术突
破。一个是将荧光分子标记在磷酸上，这样在反应停止且捕获荧光信号以后，
可直接随磷酸基团脱落，解决了因噪音污染导致的读长很短的问题；二是由于
不需要PCR扩增，信号的有效提取成为了关键。通过引入零模波导孔（ZMW）
技术解决这一问题。在纳米室底部有一个孔径70nm的小孔，由于远远小于激光
的波长，所以激光从底部照射时，只会照亮一个小的区域，提高了信噪比。
作者：生信空间
链接：https://www.jianshu.com/p/c9ade91acced
来源：简书
著作权归作者所有。商业转载请联系作者获得授权，非商业转载请注明出处。

117. 第四代测序技术
纳米孔测序技术是单分子实时测序的新一代技术，主要是通过ssDNA或RNA模板
分子通过纳米孔而带来的“电信号”变化推测碱基组成进行实时测序。
基本原理：当纳米孔充满导电液时，两端加上一定电压，分子模板通过纳米孔
生成可测量电流。纳米孔的直径只能容纳一个核苷酸，单链模板就会在电场作
用下依次通过纳米孔而引起电流强度变化，通过检测相应的电流峰判断碱基，
实现实时测序。
作者：生信空间
链接：https://www.jianshu.com/p/c9ade91acced
来源：简书
著作权归作者所有。商业转载请联系作者获得授权，非商业转载请注明出处。

119. 四大测序技术的优缺点：
Sanger法测序读长长、准确度高，但是通量不高；
Illumina测序读长短、通量高、准确度高，在进行基因组组装或者结构变异
分析的时候没有优势，可用作三四代测序read的纠错；
Pacbio测序读长长、通量高、准确度不高，但可通过测序深度弥补，GC偏
差低，可进行甲基化的直接测序。
Nanopore测序读长长、通量高、准确度低，不可通过测序深度弥补，但可
通过Illumina read 纠错。
第一、二、三代测序技术都是基于边合成边测序的原理，因此Nanopore技
术被一些人称为第四代测序技术；
随着测序技术的发展和成熟，逐渐形成基因测序产业链。
作者：生信空间
链接：https://www.jianshu.com/p/c9ade91acced
来源：简书
著作权归作者所有。商业转载请联系作者获得授权，非商业转载请注明出处

120. Genetic mapping

121. Genetic mapping of a genome
 Genetic map is a representation of the genetic distance
separating genes derived from the frequency of genetic
recombination between the genes
 Genetic mapping is the process of locating genes to
chromosomes and assigning their relative genetic distances
from other known genes
Genetic crosses
Pedigree analysis

122. Some DNA sequence changes that produce a
restriction fragment length polymorphism (RFLP)

123. Genetic linkage analysis using an RFLP marker

124. Pedigree

125. Physical mapping of a genome
 Physical map is a map of genetic markers made by
analyzing genomic DNA directly, to supplement the
genetic map
Cytogenetic map: chromosomal banding patterns
FISH (fluorescent in situ hybridization) map
Restriction map
Radiation hybrid maps
Clone contig maps

126. G-Banded
Karyotype
Cytogenetic map: chromosomal banding patterns

127. FISH (fluorescent in situ hybridization) map
An example of the
results of FISH in
which fluorescently
tagged DNA probes
were hybridized to
human metaphase
chromosomes

128. Restriction map
Using rescriction enzymes that cut rarely.
Such as NotI, recognition sequence:
5’-GCGGCCGC-3’
3’-CGCCGGCG-5’
For a genome with 50% GC content, this enzyme would cut on
average once every 48 = 65,536 bp. There is a 1 in 4 chance
of G in the first position, a 1 in 4 chance of a C in the second
position, and so on.

129. Making a radiation hybrid
Human cells are irradiated by X rays
to fragment the chromosomes. The
cells are killed, but the chromosome
fragments are rescued by fusing the
irradiated cells with rodent cells.
The chromosome fragments
become integrated into the rodent
chromosomes.
Radiation hybrid maps
The closer two markers are, the
greater the probability those markers
will be on the same DNA fragment
and therefore end up in the same RH.

130. Clone contig maps
A representative YAC contig map assembled by STS mapping
STS:
Sequence-tagged site

131. The direct shotgun approach to obtaining the genomic DNA sequence
of an organism

132. Genomic Analysis

133. Genomics is the development and
application of new mapping, sequencing,
and computational procedures for the
analysis of the entire genome of organism.
Structural genomics
Functional genomics
Comparative genomics

134. Mapping approach
Genetic mapping of a genome
Genetic markers for genetic mapping experiments
Restriction fragment length polymorphisms (RFLPs)
Variable number of tandem repeats (VNTRs)
short tandem repeats (STRs)
Single nucleotide polymorphisms (SNPs)

135. DNA microarray

137. Making microarray by synthesis
Affymetrix公司率先开发了寡聚核苷酸原位光刻技术，并申请相关专利。它是
生产高密度寡核苷酸基因芯片的核心关键技术。该技术的最大优点在于用很少
的步骤可合成大量的DNA阵列（主要步骤：固基羟基化-光敏基团保护-避光膜
透光聚合-光照活化-碱基结合-反应合成-不断循环）

138. Preparing a GeneChip®

139. Illustration of an experiment using a GeneChip®

140. Comparison of DNA Chip Technologies
Sensitivity of DNA chip based assays is a function of:
 Probe and target DNA/RNA (Complexity)
 Chip surface (autofluorescence & non-spec. bkg)
 Attachment chemistry/methodology (hyb. efficiency &
crosshyb.)
 Hybridization efficiency (lots of factors)
 Detection technology (signal type, efficiency, noise)
Oligo-Chip cDNA-Chip Genomic Chip
8 n or 20 n < 2,000 n > 50,000 n
sequencing expression expression genomic analysis

141. Tiling arrary

142. Scanned image

147. Other microarray

148. Antibody microarray

149. ELISA (enzyme linked
immunosorbent assay)

150. Frontiers in Biology 2012, Vol. 7 i ssue (4):
336-349 https://doi.org/10.1007/s11515-012-
1236-9
Functional protein microarray:
an ideal platform for
investigating protein binding
property