Section Section I – Gene libraries and I – Gene libraries and screeningscreening

I1 Genomic librariesI1 Genomic libraries Representative gene libraries, Size of library,

Genomic DNA, Vectors

I2 cDNA librariesI2 cDNA libraries mRNA isolation, purification and fractionation, Sy

nthesis of cDNA, Treatment of cDNA, Ligation to vector

I3 Screening proceduresI3 Screening procedures Screening, Colony and plaque hybridization, Expr

ession screening, Hybrid arrest and release, Chromosome walking

ContentsContents

I1 Genomic libraries — I1 Genomic libraries —

Representative gene librariesRepresentative gene libraries

• Genomic library: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 librariesGenomic libraries

cDNA libraries cDNA libraries

Gene library (made from genomic DNA)

(made from cDNA- copy of mRNA)

Important consideration:

Making a representative library---

Containing all the original sequences

(1) Lacking restriction sites

(2) Does not contain sufficient clones

(3) Enrich certain sequences, lack others

Missing original sequence:Missing original sequence:

Too long for the vector used

I1 Genomic libraries — I1 Genomic libraries — Size of librarySize 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 5-10kb, as only a few thousand recombinants will be needed.

I1 Genomic libraries — I1 Genomic libraries — Genomic DNAGenomic DNA

Purify genomic DNA

Correct size for cloning into the chosen vector:Physical shearing and restriction enzyme digestion

EukaryotesEukaryotes

ProkaryotesProkaryotes

Clone the fragments into vectors

1. Purification of genomic DNA

Remove protein, lipids and other unwanted macro-molecules by protease digestion and phase extraction（ Phenol-chloroform） .

Eukaryotes:Eukaryotes: Prepare cell nuclei (fractionation, reduce contamination from organelle DNA)

Prokaryotes: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 — I1 Genomic libraries — VectorsVectors

According to genome’s size, select a proper vector to construct a library .

Vectors Plasmid phageλ cosmid YAC

insert (kb) 10 23 45 1000

λ replacement vectorλ replacement vector

2.Ligation

3. Packing with a mixture of the phage coat proteins and phage DNA-processing enzymes

4. Infection and formation of plaques

1.Preparation of arms and genomic inserts

Library constructed

I2 cDNA libraries — I2 cDNA libraries — mRNA isolation, mRNA isolation, purification and fractionation purification and fractionation

• The most commonly chosen genomic cloning vectors are λ rep

lacement vectors which must be digested with restriction enzy

mes to produce the two λ end fragment or λ arms between whic

h 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

mRNA isolation, purification

Check the RNA integrity

Fractionate and enrich mRNA

Synthesis of cDNA

Treatment of cDNA ends

Ligation to vector

2. Methods to isolate mRNA

• Most eukaryotic mRNAs are polyadenylated at their 3’ ends.

• oligo (dT) can be bound to the poly(A) tail and u

sed to recover the mRNA.

3’-AAAAAAAAAAn5’- cap

(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 m

agnetic beads directly to a cell lysate and ‘pulling ou

t’ the mRNA using a strong magnet.

(3)Lying cells and then preparing mRNA-ribosome com

plexes on sucrose gradients.

Three methods:

100mM NaCl

rRNA and tRNA

10mM Tris, 1mM EDTA, poly(A) and -oligo(dT)

Make sure that the mRNA is not degraded.

Methods:(1)Translating the mRNA : use cell-free translation syste

m 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

3. Check the mRNA integrity

4. Cloning the particular mRNAs

Is useful especially one is trying to clone a particul

ar 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 — I2 cDNA libraries — Synthesis of cDNASynthesis of cDNA

• The first strand and Second strand synthesis

I2 cDNA libraries — I2 cDNA libraries — Treatment of cDNATreatment of cDNA

• Blunt end ligation of large fragments is not efficient, so we have to use special linkers to create sticky ends for cloning.

DNA linker:

DNA adaptor:

HO-CCGAATTCGGGGGG 3’-GGCTTAAGCCCCCC

HO - CGGGGGG 3’-TTAAGCCCCCC

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 — I2 cDNA libraries — Ligation to vectorLigation to vector

Any vectors with an EcoRI site would suitablefor cloning the cDNA.

The process : Dephosphorylate the vector

Ligate vector and cDNA with T4 DNA ligase Plasmid or 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 — I3 Screening procedures — ScreeningScreening

• 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 pr

otein 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 — I3 Screening procedures —

Colony and plaque hybridizationColony 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)

Wash to remove unhybri-dized probe and visualize

X-ray film (radio-actively labeled )

Antibody or enzyme(modified nucleotide la

beled)

Line up the hybridizated region orrepeated hybridization

Transfer to nitrocelluloseor nylon membrane

Denature DNA (NaOH). Bake onto membrane

Probe with 32p-labled DNA complementary to gene of interest

Expose to film

Select positive from master plate

Keep master Plate

Screening by plaque hybridization

I3 Screening procedures — I3 Screening procedures —

Expression screeningExpression screening• If the inserts are cloned into an expression si

tes, 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 — I3 Screening procedures —

Hybrid arrest and releaseHybrid arrest and release

• (1) Hybrid arrested translation• Individual cDNA clones or pools of clones can be used t

o hybridize to mRNA preparation.• Translate the mRNA population directly, and the inhi

bition of translation of some products detected.

• （ 2 ） Hybrid release translation• Purify the hybrids and the hybridized mRNAs release

d from them and translated, it identifies the protein encoded by the cDNA clone

I3 Screening procedures — I3 Screening procedures —

Chromosome walkingChromosome walking

Definition: To clone the desired gene by repe

ated isolating adjacent genomic clones from

the library.

Multiple choice Multiple choice questionsquestions

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 orga

nism. C genomic libraries must contain a minimum number of recombinants if they are to cont

ain 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 t

o vector. B mRNA preparation cDNA synthesis using reverse transcriptase second strand synthes

is using terminal transferase, ligation to vector. C mRNA synthesis using RNA polymerase reverse transcription of mRNA, second stran

d synthe sis, ligation to vector. D double stranded cDNA synthesis restriction enzyme digestion addition of linkers ligatio

n 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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