section i: gene libraries and screeningyang xu, college of life sciences section i gene libraries...

Section I: Gene libraries and screening Yang Xu, College of Life Sciences

Section I Gene libraries and screening I 1 Genomic libraries I 2 cDNA Libraries I 3 Screening Procedures


I 1 Genomic libraries

• Gene libraries

• Size of libraries

• Genomic DNA

• Vectors


Gene libraries

Gene library: It is a collection of different DNA sequences from an organism, each of which has been cloned into a vector for ease of purification, storage and analysis.

(1) Genomic libraries: Gene libraries made from genomic DNA;

(2) cDNA libraries: If the DNA is a copy of an mRNA population, that is cDNA, then the gene library is called a cDNA library.

Structure of representative gene libraries:

• Containing all the original sequences;

• Containing the necessary restriction sites;

• Containing a sufficient number of clones, otherwise some genes will be missing.

Gene library

Genomic libraries

cDNA libraries


Size of libraries• Recombinant number: A gene library must contain a certain

number of recombinants, so as to have a high probability of any particular sequence.

• The recombinant (transformants) number (N): can be calculated if the genome size and the average size of the insert in the vector are known. The formula is:

N = ln (1-P) / ln (1-f) where P is the desired probability and f is the fraction of the

genome in one insert.• For example: desired probability is 0.99; insert sizes is 20

kb and the size for the E.coli is 4600 kb.N = ln (1-0.99) / ln [1-(20/ 4,600)] = 1100

Why it is not 4600/20=230？


Genomic DNA

Making of a representative genomic library: Genomic DNA must be purified and broken randomly into fragments with correct size for cloning into the chosen vector.

Purification: • Prokaryotes can be extracted by phase extraction; • Eukaryotes first preparing cell nuclei and

then removing unwanted proteins and lipids by protease digestion and phase extraction.

Breaking: There are physical shearing and restriction digestion.• Physical shearing: SonicationSonication will break the DNA into smaller

fragments. • Partial digestion: to generate genomic DNA fragments of 15-25 kb

(a convenient size for and cosmid vectors). Example: Sau3A cleaves to produce a sticky end that is compatible with a vector that has been cut with BamH1


Vectors for Genomic Libraries

Vectors: Plasmids, phage, cosmid, YAC or BAC vectors can be used to construct genomic libraries. The choice depends on the genome size.

Segment size: The upper size limit of these vectors is about 10, 23, 45, 1000 and 350 kb respectively.

Plasmids

10 kb

phage

23kb

cosmid

45kb

YAC

1000 kb

BAC

350kb


I 2 cDNA libraries

1. mRNA isolation, purification and fractionation

2. Synthesis of cDNA

3. Treatment of cDNA ends

4. Ligation to vector


mRNA isolation and purificationmRNA source for cDNA library making: • They are not made from prokaryotic mRNA, since it is

very unstable;

• Making cDNA libraries from eukaryotic mRNA is very useful because cDNAs have no intron and can be expressed into proteins in E. coli.

mRNA isolation and purification: • Total RNA: by extracting lysed cells with phenol-

chloroform;• Isolating mRNA: Oligo(dT) can be bound to the poly

(A) tail of mRNA and used to recover the mRNA.


mRNA isolation and purification

• Isolating mRNA: Oligo(dT) can be bound to the poly (A) tail of mRNA and used to recover the mRNA.

– Traditional method: This was done by passing a preparation of total RNA down a column of oligo (dT)-cellulose;

– New Method: To keep damage by nuclease to a minimum, the more rapid procedure of adding oligo(dT) linked to magnetic beads directly to a cell lysate and “pulling out” the mRNA using a strong magnet.


Checking the integrity of an mRNA: Before using an mRNA preparation for cDNA cloning, it is advisable to check that it is not degraded. Main two methods are as below:

• Translation: See if the protein expression.– Cell-free translation systems: such as wheat germ extract or

rabbit reticulocyte lysate;– Microinject: to put the mRNA preparations into cells to

check whether their proteins are translated.• Gel electrophoresis: It is also a common way.

– The mRNA preparation: usually produces a smear of molecules from about 0.5 kb up to about 10 kb or more;

– The two largest rRNA: contaminate the mRNA preparations because of their abundance and appear as clear bands within the smear of mRNA.

mRNA function check


Subtracted cDNA library: to make a cDNA library of all the mRNA sequences just in the induced cells, one would prepare mRNA from both induced and non-induced cells.

• ssDNA: are made from the non-induced cell mRNA;

• mRNA: are extracted from the induced cells; • [ssDNA/mRNA]: the complementary

sequences of both ssDNA and mRNA hybridize each other to form duplexes.

• The rest mRNA: is newly induced mRNA, which could then be isolated (e.g. using magnetic beads) for constructing subtracted cDNA library.

Subtracted cDNA library


Synthesis of cDNASynthesis of cDNA

The first DNA strand making: – A primer to extend the

mRNA template. This primer is usually oligo(dT);

– All four dNTPs and reverse transcriptase must be added;

– Adding the terminal transferase and dCTP;

– Alkali for hydrolyzes RNA and purify the DNA;The second DNA strand

making:– Adding reverse transcriptase

(or Klenow polymerase) and four dNTPs.

AAAAA-3’5’mRNA

HO-TTTTTp-5’RT trans.4 dNTP

AAAAA-3’5’mRNA

TTTTTp-5’

3’cDNA

AAAAA-3’5’mRNA

3’cDNA

TTTTTp-5’

terminal transferase

CCC-3’ 3’-CCCCCCCC

Alkali for RNAAlkali for RNA

3’cDNA

TTTTTp-5’

3’-CCCCCCCC3’ -3’5’-pGGGG

3’cDNA

TTTTTp-5’

3’-CCCCCCCC5’-pGGGG-OH


Treatment of cDNA endsSolution: Blunt end ligation of large fragments is not efficient, usually, special

nucleic acid linkers are added to create sticky ends for cloning. 3’ -3’5’-pGGGG3’ TTTTTp-

5’ 3’-CCCCCCCC

1. 3’ -3’5’-pGGGG3’ TTTTTp-

5’ 3’-CCC

2.3’ -3’5’-pGGGG3’ TTTTTp-

5’ 3’-CCCC

3. 3’ -3’5’-pGGGG3’ TTTTTp-

5’ 3’-CCCC

CCGAATTCGG-3’GGCTTAAGCC-OH

HO-CCCAATTCGGGGGG3’-GGCTTAAGCCCCCC

3’ -3’5’-pGGGG3’ TTTTTp-

5’ 3’-CCCC

5’-pAATTCGGGGGG3’-GCCCCCC

CCG-3’GGCTTAAp-5’

4.


Ligation to vector Ligation to vector: Any vector

with an EcoRI site will be suitable for cloning the cDNA.

• Dephosphorylate: It is usual to dephosphorylate the vector with the enzyme alkaline phosphatase as this will prevent the vector fragments (ends) rejoining during ligation.

• Ligation: It is carried out using T4 DNA ligase, and the reeombmant molecules are either packaged (see Topic H2) or transformed (see Topic G2) to create the cDNA library.

HO -AATTC

HO -G

G -OH

CTTAA -OH

E E

E ES

PP

G -OH

CTTAA -OH

HO -AATTC

HO -G


I 3 Screening Procedure

• Screening for libraries

• Colony and plaque hybridization

• Expression screening

• Hybrid arrest and release


Screening for Libraries

Definition: The process of identifying one particular clone containing the target gene from among the very large number of others in the gene library is called the screening for libraries.

Nucleic acid probe design: The sequence mainly comes from: Some knowledge from gene bank database or papers about

the target gene may be used for making a nucleic acid probe. If sufficient of the protein product is available to permit

determination of some ammo acid sequence, which can be used to derive possible DNA sequences to make a mixture of probes.

Making of the probes: Automated chemical synthesis and then followed by PCR propagation.

Screening methods: 1. Hybridization with probe; 2. Expression screening; 3. Hybrid arrest and release translation; 4. Chromosome

walking.


Colony and plaque hybridization with probe-I

Definition: Hybridization with probe is an important method for screening a single clone which contains gene library. Although gene libraries produce plaques not colonies, after the initial step, both hybridization ways are the same.

Select positive from master plate

Keep master plate

Transfer to nitrocellulose or nylon membrane

Denature DNA (NaOH)Bake on to membrane

Probe with 32P-labeledDNA complementary toGene of interest

Expose to film


Colony and plaque hybridization with probe-II

1. Transferring: Some of the DNA in the plaque or colony can be transferred to a nylon or nitrocellulose membrane, after which are placed on top of the master plate.

2. Replica plating: Growing a colony replica on another dish directly on the membrane surface.

3. Lysing: The bacteria on the membrane are lysed by soaking in SDS and a protease, so that the colonies release DNA.

4. Denaturing and baking: The DNA on the membrane is then denatured with alkali (NaOH) to produce single strands, which are bonded to the membrane by baking or UV irradiation.


5. Hybridization: The membrane is then immersed in a solution containing a nucleic acid probe, which is usually radioactive (32P-labeled) and incubated to allow the probe to hybridize the target gene. And then, the membrane is washed to remove unhybridized probe.

Colony and plaque hybridization with probe-III

6. Auto-radiography: The regions hybridized of the membrane are then visualized by the exposure to X-ray film.

7. Comparing: By comparing the membrane with the master plate, the original group of colonies or plaques can be identified.

8. Isolating: The master plate is kept to isolate the corresponding clone and the recombinant phage.

9. Re-plating: This group is re-plated at a lower density, and the hybridization process repeated until every single individual clone is isolated.


Expression screening-IDefinition: If specific antibodies had been raised to the proteins,

the specific binding to the proteins can be used for screening the cDNA libraries that express special proteins.

Mechanism: • Cloning: By cloning cDNAs into the EcoRI site of gtll, there

is a one in six chance that the cDNA will be in both the correct orientation and reading frame to be translated into its product.

• Expressing: The EcoRI site is near the C terminus of the lacZ gene, and the insert must be linked to that of lacZ, in the correct orientation and frame, for expressing a -gal fusion protein containing the cDNA gene product.

• Screening: The -gal fusion protein may contain regions of polypeptide (epitopes) that will be recognized by antibodies raised to the native protein. These antibodies can therefore be used for screening the expression library.


Expression screening-II

Procedure: 1. Transferring: It has similarities to the plaque hybridization

protocol (above), though in this case it is the protein encoded by the cDNA rather than the DNA itself that is transferred on the membrane.

2. Binding: The membrane is treated to covalently attach the protein, and immersed in a solution of the antibody. When the antibody has bound to its epitope, it is detected by other antibodies and/or chemicals that recognize it.

3. Comparing: By comparing the membrane with the master plate, the location of the plaque can be narrowed down.

4. Isolating: Repeat cycles of screening are again required to isolate pure plaques.


Hybrid arrestHybrid arrested translation:

cDNAhybridized mRNA proteins • Hybridization: Individual cDNA clones

or pools of clones can be hybridized to mRNA preparations. The hybridized mRNA can not be translated to proteins, and the detection of proteins can be used to find which cDNA can arrest which protein.

• Subdivision: of the pools of cDNA into smaller numbers and repeating the experiment should allow the identification of a single cDNA that arrests the translation of one protein.

mRNA

cDNA


Hybrid release

Hybrid release translation: cDNAhybridized mRNAproteins

• Purification: After hybridization, the hybrids can be purified, e.g. the [cDNA/mRNA] are attached to magnetic beads or are precipitable with an antibody on membrance.

• Release: Then the mRNAs are released from hybrids (by heat or denaturant) and translated. This hybrid release translation identifies which cDNA clone encode which protein.

mRNA

mRNA

cDNA


That’s all for Section I

section i: gene libraries and screeningyang xu, college of life sciences section i gene libraries...

Documents