Transcript cDNA libraries I

Molecular Biology Course
Gene libraries and screening
（基因文库与筛选）
Gene libraries and screening
I1 Genomic libraries
（基因组文库）
I2 cDNA libraries
（ cDNA 文库）
I3 Screening procedures
（筛选流程）
Gene libraries and screening
I1 Genomic libraries
I1-1
I1-2
I1-3
I1-4
Representative gene libraries
Size of library
Genomic DNA
Vectors
I1 Genomic libraries
Gene libraries： 来自某种生物的不同DNA序列
的总集，这些DNA序列已经被克隆在载体上，以便于
纯化、贮存和分析。
Genomic libraries （基因组文库）
Gene libraries
(made from genomic DNA)
(根据来源不同)
cDNA libraries
（cDNA文库）
(made from cDNA- copy of mRNA)
I1 Genomic libraries
I1-1 Representative gene libraries
--- Contain all the original sequences
Missing original sequence
1. Certain sequences have not been cloned.
2. Library does not contain sufficient clones.
I1 Genomic libraries
I1-2 Size of library (ensure enough clones)
The formula to calculate the number of
recombinants:
ln (1-P)
N=
ln (1-f)
P: desired probability
f : the fraction of the genome in one insert
I1 Genomic libraries
For example :for a probability of 0.99 with
insert sizes of 20 kb these values for the Ecoli
(4.6×106 bp) and human (3×109 bp) genomes
are :
N Ecoli =
Nhuman =
ln( 1-0.99)
ln[1-(2×104/4.6×106)]
ln(1-0.99)
ln[1-(2 ×104/3 ×109)]
= 1.1 ×103
= 6.9 ×105
These values explain why it is possible to make good
genomic libraries from prokaryotes in plasmids where
the insert size is 5-10kb ,as only a few thousand
recombinants will be needed.
I1 Genomic libraries
I1-3 Genomic DNA libraries
Eukaryotes
真核生物
Purify genomic DNA
(纯化基因组DNA)
Prokaryotes 原核生物
Break DNA into fragments randomly
(随机切割DNA)
Clone these fragments into vectors
（将片段克隆入载体）
I1 Genomic libraries
Purification of genomic DNA :
Eukaryotes
真核细胞采用细
胞分级分离法
Prokaryotes
prepare cell nuclei
remove protein, lipids and
other unwanted macromolecules by protease
digestion and phase extraction.
extracted DNA directly from cells
I1 Genomic libraries
Break DNA into fragments randomly:
Physical shearing （物理剪切）
pipeting, mixing or sonication
（吹打、振荡、超声波）
Restriction enzyme digestion（限制性酶解）
partial digestion is preferred to get a greater
lengths of DNA fragments.
I1 Genomic libraries
Selection of restriction enzyme
1. 酶切产生的末端是否能和载体直接相连？
2. 酶的作用是否被DNA碱基修饰作用所抑制？
3. 酶解的时间影响最后产生的目的片段的大小？
I1 Genomic libraries
I1-4 Vectors
根据基因组的大小来选择合适的克隆载体
Vectors
insert (kb)
Plasmid
5
phageλ
23
cosmid
45
YAC
1000
常用的基因组克隆载体 λ relacement vectors（替换型）
I 2 cDNA libraries
I 2 cDNA libraries
1.No cDNA library was made
from prokaryotic mRNA.
• 原核的 mRNA 不稳定
• 原核的基因组文库更容易制备，并且可以包含
所有的基因组序列
I 2 cDNA libraries
cDNA libraries
2.cDNA libraries are very useful
for eukaryotic gene analysis
•
•
•
•
cDNA从mRNA逆转录而来，代表了基因中的可转录部
分（去除了非转录序列）
cDNAs 不含有内含子 （intron sequences ），可以
直接在大肠杆菌中表达
可以用于鉴定新基因
不同类型的细胞和组织表达特定的基因（奢侈基因）
Gene libraries and screening
cDNA libraries
mRNA isolation、purification
mRNA的提取、纯化
Check the RNA integrity
检测mRNA的完整性
Fractionate and enrich mRNA
mRNA的分级分离、富集
Synthesis of cDNA
合成cDNA
Treatment of cDNA ends
cDNA的末端处理
Ligation to vector
与载体连接
I 2 cDNA libraries
I2-1 mRNA isolation
• Most eukaryotic mRNAs are polyadenylated at
their 3’ ends
5’ cap
3’ AAAAAAAAAAn
• oligo (dT) can be bound to the poly(A) tail
and used to recover the mRNA.
I2 cDNA libraries
Three methods to isolate mRNA
1.传统的提取方法是把总 RNA 通过寡聚 (dT)-纤维素
柱。
2.直接将结合了寡聚 (dT)的磁珠加入到细胞裂解液中，
利用磁性将吸附了mRNA的磁珠分离出来后，再用溶
剂把mRNA从磁珠上洗脱。
3. 利用蔗糖梯度，从细胞裂解液中制备mRNA－核
糖体复合物，再提取mRNA。
I2 cDNA libraries
I2 cDNA libraries
I2-2 Check the mRNA integrity
Make sure that the mRNA is not degraded.
（确定mRNA没有被降解）
Methods:
Translating the mRNA
（翻译mRNA）
Analysis the mRNAs by gel elctrophoresis
（琼脂糖凝胶电泳或聚丙烯酰胺凝胶电泳）
I2 cDNA libraries
I2-3 Cloning the particular mRNAs
为了克隆某一特定基因，而不是构建完整的cDNA
文库，需要对mRNA进行分级分离或者富集。
Fractionate on the gel
（利用mRNA的大小不同，从凝胶中回收）
Subtracted cDNA library
(扣除 cDNA 文库)
I2 cDNA libraries
I2-4 Synthesis of cDNA
First stand synthesis
reverse transcriptase(反转录酶) 、primeroligo(dT)（寡聚dT引物） 、dNTPs（4种）
Second strand synthesis
对第一链的3’端“加尾”， 再用与之互补的引物合成
第二链，保证获得全长cDNA。(Fig 1.1)
I2 cDNA libraries
5’
5’
3’
mRNA
AAAAA-3’
Reverse transcriptase HO-TTTTTP-5’
4 dNTPs
primer
mRNA
AAAAA-3’
TTTTTP-5’
Terminal transferase（末端转移酶）
dCTP
mRNA
AAAAACC-3’
TTTTTP-5’
cDNA
cDNA
5’
3’-CCCCCCC
primer
Alkali (hydrolyaes RNA)
Purify DNA oligo(dG)
5’-pGGGG-OH
3’-CCCCCCC
cDNA
5’-pGGGG
3’-CCCCCCC
TTTTTP-5’
Klenow polymerase or reverse
Transcriptase 、4dNTPs
Duplex cDNA
-3’
TTTTTP-5’
Fig 1.1 The first and second strands synthesis
I2 cDNA libraries
I2-5 Treatment of cDNA ends
大片段的平末端连接效率很低，所以必需对双链cDNA的末端
进行加工，可以通过添加接头（linkers）进行改造。
The process :
切除突出的 3’-ends (单链特异性核酸酶)
补平3’-ends (DNA聚合酶的klenow 片段和 dNTPs)
连接双链cDNA的平末端和接头 (T4 DNA ligase)
接头中可以嵌入酶切位点(EcoR I )
酶切产生粘性末端 (EcoR I )
5’-pGGGG
3’-CCCCCCC
Duplex cDNA
5’-pGGGG
3’-CCC
-3’
TTTTTp-5’
单链特异性核酸酶
-3’
TTTTTp-5’
Klenow 聚合酶、dNTP
用 EcoR I 甲基化酶处理
5’-pGGGG
3’-CCCC
加入嵌入酶切位点的
接头进行连接
-3’
TTTTTp-5’
HO-CCG/AATTCGG-3’
3’-GGCTTAA/GCC-OH
接头
CCG/AATTCGG-3’
TTTTTGGCTTAA/GCC-OH
HO-CCG/AATTCGGGGGG
3’-GGCTTAA/GCCCCCC
EcoR I 消化
5’-pAATTCGGGGGG
3’-GCCCCCC
CCG-3’
TTTTTGGCTTAAp-5’
Ligate to vector and transform
Fig2.1 end preparation and linker addition to duplex cDNA
I2 cDNA libraries
I2-6 Ligation to vector（载体）
Any vectors with an EcoR I site would suitable
for cloning the cDNA.
The process :
载体去磷酸化（避免自连）
连接载体和cDNA片段（ T4 DNA ligase）
(plasmid or λ phage vector)
Gene libraries and screening
I3 Screening procedures
I3-1
Screening（筛选）
I3-2 Colony and plaque hybridization
（菌落及噬菌斑杂交）
I3-3 Expression screening
（表达筛选）
I3-4 Hybrid arrest and release
（杂交扣留与释放）
I3-5 Chromosome walking (repeat
screening)（染色体步移）
I3 Screening procedures
I3-1 Screening
Screening：从基因文库的大量克隆中鉴定出某个含
有目的基因的特定克隆的过程。
1. Using nucleic acid probe（核酸探针） to screen
the library based on hybridization（杂交） with
nucleic acids.
2. Analyze the protein product.
I3 Screening procedures
Screening libraries
Searching the genes of interest in a DNA library
Hybridization to identify the interested DNA or
its RNA product
1. Radiolabeled probes （放射性标记的探针）
2. Blotting the DNA or RNA on a membrane
（将目的DNA或RNA吸附在膜上）
3. Hybridize the labeled probe with DNA membrane
(Southern) or RNA (Northern) membrane
（加入探针，在膜上进行杂交）
I3 Screening procedures
I3-2 Colony and plaque hybridization
Transfer the DNA in the plaque or colony to a
Nylon or nitrocellulose membrane
Bacterial colonies must be lysed to
Phage DNA bind to
release DNA on the membrane
the membrane directly
surface.
（噬菌斑）
(菌落，碱裂解)
Hybridization (in a solution
Containing Nucleic acid probe)（与探针杂交）
X-ray
film(radioactively
labeled )
Wash to remove unhybridization probe and visualize
antibody or
enzyme
(抗体或酶）
Line up the hybridizated region or
repeated hybridization (标出被杂交区域，重复杂交)
I3 Screening procedures
Transfer to nitrocellulose
or nylon membrane
Keep master
plate
Select positive
from master plate
Denature DNA(NaOH)
Bake onto membrane
Probe with 32p-labled DNA
complementary to
gene of interest
Expose to film
Screening by plaque hybridization
I3 Screening procedures
I3-3 Expression screening
（表达筛选）
Identify the protein product of an
interested gene
1. Protein activity
2. Western blotting using a specific
antibody
I3 Screening procedures
Protein
activity
If the inserts are cloned into an expression
sites, it may be expressed. Therefore, we can
screen for the expressed proteins. （However, this
screening may miss the right clone.）
Example: the EcoR I site of lgt11 vector. The
inserted genes have one in six change (1/6) to
be in both the correct orientation (2
possibilities;  ) and reading frame (three
possibilities; three nucleotide code XXX).
I3 Screening procedures
Antibodies （抗体）can be used to screen the
expression library. The procedure has similarities to
the plaque hybridization（噬菌斑杂交） protocol.
噬菌斑复制
‘Plaque lift’ ( taken by placing a
membrane on the dish of plaque)
抗体结合
Immersed in a solution of the antibody
抗体识别
Detected by other antibodies
重复循环
筛选
Repeat cycles of screening to isolate
pure plaques
I3 Screening procedures
I3-4 Hybrid arrest and release
(杂交扣留与释放）
Individual cDNA clones or pools of
clones can be used to hybridize to
mRNA preparation.
单个cDNA克隆或克隆群都可以用来与
mRNA样品进行杂交。
Hybrid arrest :translate the mRNA population
directly, and the inhibition of translation of
some products detected.
Hybrid release translation : purify the
hybrids and the hybridized mRNAs released
from them and translated, it identifies the
protein encoded by the cDNA clone
I3 Screening procedures
I3-5 Chromosome walking
（染色体步移）
Definition: To clone the desired gene by
repeated isolating adjacent
genomic clones from the library.
to obtain overlapping genomic clones
that represent progressively longer
parts of a particular chromosome .
I3 Screening procedures
Process:
1. Prepare a probe from the end insert .
2.The probe are used to re-screen the library
by colony or plaque hybridization
3.Analyzed the new isolate clones and posited
them relative to the starting clone.
some will be overlapping.
4. Repeated the whole process using a probe
from the distal end of the second clone.
Vector arm
}
}
Restriction
Genomic clone insert
Vector arm
Prepare probe from
ends of insert
Re-screen genomic
library
Restriction map new
genomic clones
}
}
Prepare new probes from distal ends of least overlapping insert.
Re-screen genomic library . Restriction map new genomic clones
Chromosome walking
over !