Download Section D - Prokaryotic and Eukaryotic Chromosome Structure

Section I – Gene libraries and
screening
Contents
I1 Genomic libraries
Representative gene libraries, Size of library,
Genomic DNA, Vectors
I2 cDNA libraries
mRNA isolation, purification and fractionation,
Synthesis of cDNA, Treatment of cDNA, Ligation
to vector
I3 Screening procedures
Screening, Colony and plaque hybridization,
Expression screening, Hybrid arrest and release,
Chromosome walking
I1 Genomic libraries —
Representative gene libraries
• Genomic library: A collection of different DNA
sequence from an organism each of which has been
cloned into a vector for ease of purification, storage
and analysis .
Genomic libraries
Gene library
(made from genomic DNA)
cDNA libraries
(made from cDNA- copy of mRNA)
Important consideration:
Making a representative library--Containing all the original sequences
Missing original sequence:
(1) Lacking restriction sites
Too long for the vector used
(2) Does not contain sufficient clones
(3) Enrich certain sequences, lack others
I1 Genomic libraries —
Size of library
The formula to calculate the number of recombinants:
P: desired probability
f : the fraction of the genome in one insert
For a probability of 0.99 with insert sizes of 20kb
these values for the E. coli (4.6×106 bp) and human
(3×109 bp) genomes are :
Easy to make good genomic libraries from
prokaryotes in plasmids where the insert size is 510kb, as only a few thousand recombinants will be
needed.
I1 Genomic libraries —
Genomic DNA
Eukaryotes
Purify genomic DNA
Prokaryotes
Correct size for cloning into the chosen vector:
Physical shearing and restriction enzyme digestion
Clone the fragments into vectors
1. Purification of genomic DNA
Eukaryotes: Prepare cell nuclei (fractionation, reduce
contamination from organelle DNA)
Remove protein, lipids and other unwanted
macro-molecules by protease digestion and
phase extraction（Phenol-chloroform）.
Prokaryotes: Extracted DNA directly from cells
2. Break DNA into fragments randomly
(1) Physical shearing
Pipeting, mixing or sonication. The choice
of method and time of exposure depend on
the size requirement of the chosen vector.
(2)Restriction enzyme digestion
Partial digestion:
Get a greater lengths of DNA fragments. Time of
digestion and ration of restriction enzyme to DNA
are dependent on the desired insert size range, the
DNA is not digested at every recognition sequence
that is present.
Sau3A: 5’-/GATC-3’,
BamH1: 5’-G/GATCC-3’
Ends produced (sticky or blunt) &
the cleaved ends of the vector to be
cloned
DNA modifications
Whether the enzyme is inhibited by DNA
modifications (CpG methylation in
mammals).
I1 Genomic libraries —
Vectors
According to genome’s size, select a proper
vector to construct a library .
Vectors
Plasmid
insert (kb)
10
phageλ
23
cosmid
45
YAC
1000
λ replacement vector
1.Preparation of arms and
genomic inserts
2.Ligation
3. Packing with a mixture of
the phage coat proteins and
phage DNA-processing
enzymes
4. Infection and
formation of plaques
Library constructed
I2 cDNA libraries —
mRNA isolation,
purification and fractionation
• The most commonly chosen genomic cloning vectors are λ
replacement vectors which must be digested with restriction
enzymes to produce the two λ end fragment or λ arms between
which the genomic DNA will be ligated.
1. Characteristics of cDNA libraries
2. Methods to isolate mRNA
3. Check the mRNA integrity
4. Cloning the particular mRNAs
1. Characteristics of cDNA libraries
(a) No cDNA library was made from prokaryotic
mRNA.
•
•
Prokaryotic mRNA is very unstable
Genomic libraries of prokaryotes are easier to make and
contain all the genome sequences.
(b) cDNA libraries are very useful for eukaryotic
gene analysis
•
•
cDNAs represent the transcribed parts of the genome
(i.e. the genes rather than the nontranscribed DNA).
cDNAs have no introns  genes can be expressed in E.
coli directly
Tissue or cell type specific (differential expression of
genes
2. Methods to isolate mRNA
mRNA isolation, purification
Check the RNA integrity
Fractionate and enrich mRNA
Synthesis of cDNA
Treatment of cDNA ends
Ligation to vector
• 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.
Three methods:
(1)Traditional method was done by pass a preparation
of total RNA down a column of oligo (dT)-cellulose.
(2)More rapid procedure is to add oligo(dT) linked to
magnetic beads directly to a cell lysate and ‘pulling
out’ the mRNA using a strong magnet.
(3)Lying cells and then preparing mRNA-ribosome
complexes on sucrose gradients.
100mM NaCl
rRNA and tRNA
10mM Tris, 1mM
EDTA, poly(A) and
-oligo(dT)
3. Check the mRNA integrity
Make sure that the mRNA is not degraded.
Methods:
(1)Translating the mRNA : use cell-free translation
system as wheat germ extract or rabbit reticulocyte
lysate to see if the mRNAs can be translated
(2)Analysis the mRNAs by gel electrophoresis: Use
agarose or polyacrylamide gels
4. Cloning the particular mRNAs
Is useful especially one is trying to clone a
particular gene rather to make a complete cDNA
library
• Fractionate on the gel: Performed on the basis of size, mRNAs
of the interested sizes are recovered from agarose gels
•Enrichment: Carried out by hybridization.
•Example: make a cDNA library of the mRNA sequences that are
induced with a hormone (hybridization ， substrated cDNA
library)
I2 cDNA libraries —
Synthesis of cDNA
• The first strand and Second strand synthesis
I2 cDNA libraries —
DNA linker:
Treatment of cDNA
HO-CCGAATTCGGGGGG
3’-GGCTTAAGCCCCCC
DNA adaptor:
HO - CGGGGGG
3’-TTAAGCCCCCC
• Blunt end ligation of large fragments is not efficient,
so we have to use special linkers to create sticky
ends for cloning.
The process :
Move protruding 3’-ends( strand-special nuclease)
Fill in missing 3’ nucleotide( Klenow fragment of
DNA polyI and 4 dNTPs)
Ligate the blunt-end and linkers( T4 DNA ligase)
Restriction enzyme digestion( EcoRI )
I2 cDNA libraries —
Ligation to vector
Any vectors with an EcoRI site would suitable
for cloning the cDNA.
The process :
Dephosphorylate the vector
Ligate vector and cDNA with T4 DNA ligase
Plasmid or l phage vector, short, plasmid vector;
cDNA libraries, λ phage vector;
λgt11 has EcoRI site placed near the C terminus of its
lacZ gene, enabling expression of the cDNA as part of a
large β-galactosidase fusion protein.
I3 Screening procedures —
Screening
• Screening: The process of identifying one particular
clone containing the gene of interest from among
the
• very large number of others in the gene library .
(1) Using nucleic acid probe to screen the library
based on hybridization with nucleic acids.
(2) Analyze the protein product
Screening libraries
Hybridization to identify the interested DNA or its RNA product.
(1) DNA radiolabeled probes which is
complementary to a region of the interested gene.
DNA sequence information:
An oligonucleotide derived from the sequence of a
protein product of the gene.
A DNA fragment/oligo from a related gene of another
species.
Preparation methods:
Automated chemical synthesis (short probes)
PCR
I3 Screening procedures —
Colony and plaque hybridization
Transfer the DNA in the plaque or colony to a Nylon or
nitrocellulose membrane
Phage DNA bind to
the membrane directly
Bacterial colonies must be lysed to
release DNA on the membrane surface.
(Alkali treatment)
Hybridization (in a solution
containing Nucleic acid probe)
X-ray film (radioactively labeled )
Wash to remove unhybridized probe and visualize
Line up the hybridizated region or
repeated hybridization
Antibody or enzyme
(modified nucleotide
labeled)
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 —
Expression screening
• If the inserts are cloned into an expression
sites, it may be expressed. Therefore, we can
screen for the expressed proteins.
• Example: the EcoRI site of lgt11 vector. The
inserted genes have one in six possibilities
(1/6) to be in both the correct orientation (two
possibilities;  ) and reading frame (three
possibilities).
I3 Screening procedures —
Hybrid arrest and release
• (1) Hybrid arrested translation
• Individual cDNA clones or pools of clones can be used
to hybridize to mRNA preparation.
• Translate the mRNA population directly, and the
inhibition of translation of some products detected.
• （2）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 —
Chromosome walking
Definition: To clone the desired gene by
repeated isolating adjacent genomic clones
from the library.
Multiple choice questions
1. Which two of the following statements about genomic libraries are false?
A genomic libraries are made from cDNA.
B genomic libraries must be representative if they are to contain all the genes in an
organism.
C genomic libraries must contain a minimum number of recombinants if they are to
contain all the genes In an orgamsm.
D the DNA must be fragmented to an appropriate size for the vector that is used.
E genomic libraries made from eukaryotic DNA usually use plasmid vectors.
2. Which statement correctly describes sequential steps in cDNA cloning?
A reverse transcription of Mrna second strand synthesis cDNA end modification ligation
to vector.
B mRNA preparation cDNA synthesis using reverse transcriptase second strand
synthesis using terminal transferase, ligation to vector.
C mRNA synthesis using RNA polymerase reverse transcription of mRNA, second strand
synthesis, ligation to vector.
D double stranded cDNA synthesis restriction enzyme digestion addition of linkers
ligation to vector.
3. Which one of the following is not a valid
method of screening a library?
A hybridization of colony / plaque-lifted DNA
using a nucleic acid probe.
B using antibodies raised against the protein of
interest to screen an expression library.
C screening pools of clones from an expression
library for biological activity.
D hybridization of colony/plaque-lifted DNA using
an antibody probe.
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