Download genomic library

生物技术综合实验
Comprehensive experiments of
biotechnology
主要内容 Contents：
一、课程简介 核酸的分离与纯化 Isolation and Purification of
Nucleic Acid
二、电泳技术 Agarose Gel Elrctrophoresis and SDS-PAGE
三、聚合酶链式反应技术 Polymerase Chain Reaction
四、DNA序列测定 DNA Sequencing
五、分子杂交技术 Molecular Hybridization － Southern,Northern
and Western Blot
六、基因文库和cDNA文库的构建 Construction of cDNA Library
and DNA Library
七、外源基因的克隆与表达 Heterogenic Gene Cloning and
Expression
八、蛋白质的分离、纯化技术 Isolation and Purification of
Protein
Part 6 基因文库和cDNA文库的构建
Construction of DNA & cDNA Library
 基因组文库（genomic library）：包
含某种生物体全部基因的随机片段的
重组DNA克隆群体称为基因组文库。
 cDNA文库（cDNA library）：包含细胞
的全部mRNA的信息的重组cDNA克隆群体
称为cDNA库。
一、基因组文库（genomic library）
基因工程技术的迅速发展使人们对生物体基
因的结构、功能、表达及其调控的研究深入到
分子水平，而对特定基因片段的分离和获得是
上述研究的基础。
完整的基因组文库(genomic library)的构
建使任何DNA片段的筛选和获得成为可能。
1.1 构建真核细胞基因组文库的载体：
λ噬菌体（可插入基因片断约20kb）
粘性质粒（可插入基因片断约46kb）
酵母人工染色体克隆系统(yeast artificial
chromosome cloning system),简称YACS。
（可插入基因片断约200-500kb）
1.2 应用入噬菌体构建基因组文库的基本步骤
(1) 准备载体DNA。
(2) 提取高分子量真核细胞DNA：并选择合适
的限制性内切酶进行部分降解。
(3) 分离大小合适的真核DNA片段。
(4)载体DNA与外源DNA连接。
(5)连接产物在体外进行包装。
(6)检测重组噬菌体的滴度，扩增、分装保存。
二、cDNA文库（cDNA library）
2.1 概述
由于mRNA含有某种细胞的各种RNA分子，
因而反转录合成的cDNA将代表各样mRNA拷
贝，将其和载体DNA重组，并转化到宿主细菌
里或包装成噬菌体颗粒，得到一系列克隆群体。
每个克隆只含一种mRNA的信息，足够数目克
隆的总和则包含细胞的全部mRNA的信息，这
样的克隆群体叫cDNA库。
按照筛选方式不同cDNA库可分为：
表达型cDNA文库:采用表达型载体。插入的
cDNA片段可表达产生融合蛋白，不能采用核
苷酸探针筛选的目的基因，可采用能与表达
产物发生特异性结合的抗体或化合物进行标
记筛选。
非表达型cDNA文库：适用于那些采用核苷
酸探针进行杂交筛选的基因。
根据载体的不同将cDNA库分为：
质粒cDNA库: 包含的cDNA克隆数目较少，适于
较高丰度的mRNA
噬菌体cDNA库:包含的cDNA克隆数目非常多，
适用于那些低丰度和极低丰度的mRNA
在构建一个cDNA库时，首先应考虑的问题是
筛选方法。若采用核苷酸探针进行杂交筛选，可
构建表达型或非表达型cDNA库；如利用蛋白质的
生物活性或免疫原性进行筛选，则只能构建表达
型cDNA库。
其次应考虑的问题是mRNA的丰度。对于高丰
度的mRNA所需构建的cDNA库相对较小；而极低丰
度的mRNA，如仅占mRNA总量的1/106的某些mRNA，
所需构建的cDNA库则必须很大，尽可能包括其对
应的克隆。
2.2 构建cDNA库主要包括以下几个步骤：
① mRNA的分离；
② cDNA第一链的合成；
③ cDNA第二条链的合成；
④ cDNA与载体的连接；
⑤ 噬菌体的包装及转染或质粒的转化。
⑥ 检测重组噬菌体的滴度，扩增、分装保存。
cDNA文库构建的具体操作方法见
有关实验指南。
UNIT 2
MANIPULATION OF DNA AND GENE ISOLATION
LECTURES:
9. DNA Cloning and Library Construction
10. Isolating Genes
9. DNA Cloning and Library Construction
a). DNA cloning
i). Restriction endonucleases
ii). Cloning vectors
iii). The process of cloning a segment of DNA
b). Library construction
i). Genomic libraries
ii). cDNA libraries
DNA
mRNA
protein
How does one isolate a gene for an inherited disorder?
There are three options:
• Start with a candidate protein
DNA
protein
• Start with a candidate mRNA
DNA
mRNA
• Direct positional cloning
DNA
All three options require the cloning of DNA.
Restriction endonucleases
• Restriction enzymes cut DNA into specific fragments
• Restriction enzymes recognize specific base sequences in double-stranded
DNA and cleave both strands of the duplex at specific places
• Characteristics of restriction enzymes:
1. Cut DNA sequence-specifically
2. Bacterial enzymes; hundreds are purified and available commercially
3. Restriction-modification system
Bacteria have enzymes that will cleave foreign DNA; hence, “restrict” the entry of viral
DNA. To prevent the bacteria’s own DNA from being cut, there is a second enzyme that
methylates the same sites recognized by the restriction enzyme (modifies that site).
4. Named (e.g., EcoRI) for bacterial genus, species, strain, and type
5. Recognize specific 4-8 bp sequences
• sequences have symmetry (they are palindromes)
• after cutting the DNA, the cut ends are either
• blunt
• staggered (overhangs) - cohesive ends facilitate cloning the DNA
6. Frequency of cutting
• 4-base cutter 44 =
256 bp
• 5-base cutter 45 = 1,024 bp
• 6-base cutter 46 = 4,096 bp
• 8-base cutter 48 = 65,536 bp
4-base cutter:
cuts DNA into 256 bp average-sized fragments in a random sequence
every 256 bp: NO
256 bp average-size fragments: YES
Bar = 256 bp
Products generated by restriction enzymes
COHESIVE ENDS
EcoRI
5’…GAATTC…3’
3’…CTTAAG…5’
5’…G
3’…CTTAA
AATTC…3’
G…5’
PstI
5’…CTGCAG…3’
3’…GACGTC…5’
5’…CTGCA
3’…G
G…3’
ACGTC…5’
BLUNT ENDS
HaeIII
5’…GGCC…3’
3’…CCGG…5’
5’…GG
3’…CC
CC…3’
GG…5’
Formation of recombinant DNA molecules
cut DNAs
mix together fragments and anneal cohesive ends
seal 3’, 5’ ends by DNA ligase
recombinant DNAs
Vectors used in molecular cloning
Vector
(and host)
Plasmid
(bacteria, yeast)
Bacteriophage lambda
or phage lambda
(bacteria)
Characteristics
Insert
size range
Small circular DNA
<5 - 10 kb
Linear viral DNA
up to ~20 k
Cosmid
(bacteria)
Hybrid of plasmid
and phage
up to ~50 kb
Yeast artificial
chromosome or YAC
(yeast)
DNA containing yeast
centromere, telomeres,
and origins of replication
~200 to ~1000 kb
Structure of pBR322 - a common cloning vector
• derived from a naturally occurring plasmid
• has antibiotic resistance genes for selection of
transformants containing the plasmid
• has unique restriction enzyme cleavage sites for
insertion of foreign DNA
• has origin of DNA replication (ori) for propagation in E. coli
gene for
ampicillin
resistance
gene for
tetracycline
resistance
EcoRI
Pst I
ori
Sal I
Cloning a segment of DNA into a plasmid vector
PstI
Human DNA cut with PstI
P
ampR
P
combine
and
ligate
tetR
pBR322 ampR, tetR
P
P
tetR
pBR322 DNA cut with PstI
inactivating the ampR gene
tetR
pBR322 (human clone) tetR
• bacteria are “transformed” with the recombinant plasmid
• colonies that grow in tetracycline, but not in ampicillin are isolated
Library construction
• two types of libraries
• a genomic library contains fragments of genomic DNA (genes)
• a cDNA library contains DNA copies of cellular mRNAs
• both types are usually cloned in bacteriophage vectors
Construction of a genomic library
vector DNA (bacteriophage lambda)
Bam HI sites
“left arm”
• lambda has a linear doublestranded DNA genome
• the left and right arms are essential
for the phage replication cycle
• the internal fragment is dispensable
“right arm”
internal fragment (dispensable for phage growth)
Bam HI sites:
NNG GATCCNN
NNCCTAG GNN
cut with Bam HI
(6-base cutter)
many cells)
cut with Sau 3A (4-base cutter)
which has ends compatible
with Bam HI:
NNN GATCNNN
internal fragment
NNNCTAG NNN
remove internal
fragment
“left arm”
human genomic DNA (isolated from
“right arm”
isolate ~20 kb
fragments
“left arm”
“right arm”
combine and treat
with DNA ligase
“left arm”
2
5
3
1
6
“right arm”
package into bacteriophage
and infect E. coli
4
7
• genomic library of human DNA fragments
in which each phage contains a different
human DNA sequence
Partial restriction enzyme digestion
allows cloning of overlapping fragments
a “contig”
• isolation of ~20 kb fragments provides optimally
sized DNAs for cloning in bacteriophage
• partial digestion with a frequent-cutter (4-base cutter) allows production
of overlapping fragments, since not every site is cut
• overlapping fragments insures that all sequences in the genome are cloned
• overlapping fragments allows larger physical maps to be constructed as
contiguous chromosomal regions (contigs) are put together from
the sequence data
• number of clones needed to fully represent the human genome (3 X 109 bp)
assuming ~20 kb fragments
• theoretical minimum = ~150,000
• 99% probability that every sequence is represented = ~800,000
All possible sites:
Results of a partial digestion:
= uncut
= cut
Construction of a cDNA library
• reverse transcriptase makes a DNA copy of an RNA
The life cycle of a retrovirus depends on reverse transcriptase
retrovirus
2. the capsid is uncoated, releasing genomic
RNA and reverse transcriptase
1. virus enters cell
and looses envelope
6. it is translated into viral proteins,
and assembled into new
virus particles
new viruses
3. reverse transcriptase
makes a DNA copy
4. then copies the DNA strand to
make it double-stranded DNA,
removing the RNA with RNase H
5. the DNA is then integrated
into the host cell genome
where it is transcribed by
host RNA polymerase II
• cDNA library construction
AAAAA 3’ mRNA
5’
(all mRNAs in cell)
anneal oligo(dT) primers of 12-18 bases in length
AAAAA 3’
3’ TTTTT 5’
5’
add reverse transcriptase and dNTPs
AAAAA 3’
TTTTT 5’ cDNA
5’
3’
add RNaseH (specific for the RNA strand of an RNA-DNA
hybrid) and carry out a partial digestion
5’
3’
AA
TTTTT
short RNA fragments serve as primers for
second strand synthesis using DNA polymerase I
AAAAA
TTTTT
5’
3’
short RNA fragments serve as primers for
second strand synthesis using DNA polymerase I
AAA
TTTTT
5’
3’
DNA polymerase I removes the remaining RNA with
its 5’ to 3’ exonuclease activity and continues synthesis
AAA
TTTTT
5’
3’
DNA ligase seals the gaps
5’
3’
AAAAA double-stranded
TTTTT
cDNA
AAAAA
TTTTT
5’
3’
EcoRI linkers
NNNNNNNNG
are ligated to both ends
NNNNNNNNCTTAA
using DNA ligase
AAAAANNNNNNNNG
TTTTTNNNNNNNNCTTAA
5’
3’
AATTCNNNNNNNN
GNNNNNNNN
• double-stranded cDNA copies of mRNA with EcoRI cohesive ends are
now ready to ligate into a bacteriophage lambda vector cut with EcoRI
EcoRI sites
“left arm”
cDNAs
“right arm”
combine cDNAs with
lambda arms and treat
with DNA ligase
“left arm”
2
5
3
1
6
“right arm”
package into bacteriophage
and infect E. coli
4
7
• cDNA library in which each phage contains
a different human cDNA
cDNA Library
The Central Dogma
DNA
Precursor
RNA
mRNA
Double-stranded
exon
Single-stranded
AAAAAAAAAAn
intron
AAAAAAAAAAn
Single-stranded
Reverse transcription
Protein
AAAAAAAAAAn
double-stranded
cDNA
cDNA
A cDNA or complementary DNA, is a DNA copy of an
RNA, usually mRNA
cDNA
mRNA
Double stranded
Single stranded
Stable
unstable
Easy to manipulate
More difficult to manipulate
Need to be transcribed into
RNA to make a protein
Can be directly used to make
a protein
TTTTTTTTT
cDNA synthesis
First strand
Nick translation
cDNA library
EcoR I
Infect cells
Genomic Library
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
cDNA synthesis
cDNA library
Infect cells
Genomic library
cDNA library
Source
Genomic DNA
mRNA
Variation
Species or strains
Species or strains
Tissues
Developmental stages
Insert size
12k -- 20k
0.2k -- 6k
Representation
Equal
Correlate with
expression level
Type
Only one
Expression vs. non
expression
Probe
DNA
DNA or antibody or
protein
Purpose
Gene structure
Infer protein identity
Encoded protein
Infer protein identity
Subcloning of the cDNA insert
EcoR I
EcoR I
EcoR I
+
Is there a better way to
subclone the insert?
PCR -- polymerase chain reaction
PCR is one of the most powerful molecular biology
techniques.
It allows scientists to amplify a specific DNA region in the
test tube from extremely tiny amount of DNA sample
(even from a single molecule of DNA).
PCR -- polymerase chain reaction
Forward primer
Reverse primer
Taq DNA polymerase
5’
3’
3’
5’
1st cycle
Heat
5’
3’
3’
5’
5’
3’
5’
2nd cycle
5’
3’
3’
3’
3’
Heat
5’
3’
5’
3’
5’
5’
5’
3’
5’
3’
5’
3’
The ideal PCR products
5’
3’
3’
5’
3’
5’
3’
5’
3’
5’
5’
3’
5’
3’
5’
3rd cycle
5’
3’
3’
3’
3’
3’
Heat
5’
5’
3’
5’
3’
5’
3’
5’
5’
3’
5’
5’
5’
3’
5’
3’
3’
5’
5’
3’
5’
3’
5’
5’
3’
3’
3’
The ideal PCR
products
3’
5’
5’
3’
4th cycle
3’
5’
3’
5’
3’
5’
Heat
5’
3’
5’
3’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
5’
3’
3’
5’
3’
5’
5th cycle
Heat
6th cycle
Heat
nth cycle
Heat
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
2n products
Cloning out cDNA insert by PCR
EcoR I
PCR
EcoR I
EcoR I
+
EcoR I
EcoR I
mRNA level is very very low
“I tried very hard but could not isolate the cDNA clone
from 1 million cDNA clones”
RT-PCR -- Reverse Transcription + PCR
PCR
mRNA
cDNA
(cDNA)n
TTTTT
You need to know at least partial sequence
of the cDNA you want to amplify!
RACE method to isolate full-length cDNA
mRNA
is too
longends
Rapid
amplification
of cDNA
TTTT
Partial cDNA
Reverse transcriptase
RNase H
Terminal transferase
CCCCC
Oligo(dG)n pimer
DNA polymerase
GGGGG
CCCCC
PCR
GGGGG
CCCCC
GGGGG
CCCCC
GGGGG
CCCCC
GGGGG
CCCCC
GGGGG
CCCCC
GGGGG
CCCCC
GGGGG
CCCCC
GGGGG
CCCCC
Ligate to
CCCCC
GGGGG
Full-length cDNA
Expression of a cloned gene to study its protein functions
1) Expression of a cloned mammalian gene in mammalian cells
A genomic clone (with introns) can be used.
A cDNA (without introns) clone can be used
2) Expression of a cloned gene in a heterologous system
a. in bacteria
b. in yeast
c. in insect cells
in each case, cDNA is preferred to be used, since you
don’t know whether a mammalian gene can be spliced
correctly in a heterologous system.
cDNA
mRNA
Protein
Bacterial protein expression vectors and systems
-peptide or
N-terminus of
b-galactosiase
Multiple cloning sites
Bam HI
cDNA
BamHI
G GAT CCC ATG AGG ACC CAT AGC AAT TCG GGG CCC CCT GGG AGG GCT
Asp Pro Met Arg Thr His Ser Asn Ser Gly Pro Pro Gly Arg Ala
ATG ACC ATG ATT ACG AAT TCG AGC TCG GTA CCC GGG GAT CCC ATG AGG ACC CAT AGC AAT TCG GGG CCC CCT GGG AGG GCT
Met Thr Met Ile Thr Asn Ser Ser Ser Val Pro Gly Asp Pro Met Arg Thr His Ser Asn Ser Gly Pro Pro Gly Arg Ala
Expression vectors
To yield the product of a cloned gene for further studies
1) Expression vectors with a strong promoter
More mRNA
More protein
Expression vectors with a strong promoter
Expression vectors
To yield the product of a cloned gene for further studies
1) Expression vectors with a strong promoter
More mRNA
More protein
2) Expression vectors with an inducible promoter
Foreign proteins when overexpressed could be toxic
Keep the gene expression off till it is time to turn it on
a. Drug-inducible (e.g. IPTG or arabinose)
b. Heat-inducible
Expression vectors
To yield the product of a cloned gene for further studies
1) Expression vectors with a strong promoter
More mRNA
More protein
2) Expression vectors with an inducible promoter
Foreign proteins when overexpressed could be toxic
Keep the gene expression off till it is time to turn it on
a. Drug-inducible (e.g. IPTG or arabinose)
b. Heat-inducible
3) Expression vectors with a fusion tag for affinity purification
Facilitate the purification of the expressed protein
1) 6 Histidine tag
2) Glutathione transferase tag (GST)
3) Maltose-binding protein tag