1. Section I Gene libraries and screening. I 1 Genomic libraries
Section I Gene libraries and screening I 1 Genomic libraries I 2 cDNA Libraries I 3 Screening Procedures
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences

2. I 1 Genomic libraries Gene libraries Size of libraries Genomic DNA
Vectors
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences

3. Gene libraries Gene library
Genomic
libraries
cDNA
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.
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences

4. Size of libraries Why it is not 4600/20=230？
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？
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences

5. 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: Sonication will break the DNA into smaller fragments.
Partial digestion: to generate genomic DNA fragments of 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
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences

6. Vectors for Genomic Libraries
Plasmids
10 kb
 phage
23kb
cosmid
45kb
YAC
1000 kb
BAC
350kb
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.
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences

7. I 2 cDNA libraries Synthesis of cDNA Treatment of cDNA ends
mRNA isolation, purification and fractionation
Synthesis of cDNA
Treatment of cDNA ends
Ligation to vector
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences

8. mRNA isolation and purification
mRNA 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.
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences

9. 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.
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences

10. mRNA function check
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.
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences

11. Subtracted cDNA library
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.
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences

12. Synthesis of cDNA The first DNA strand making: mRNA
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 3’
cDNA
TTTTTp-5’
terminal transferase
AAAAA-3’ 5’
mRNA 3’
cDNA
TTTTTp-5’
CCC-3’
3’-CCCCCCCC
Alkali for RNA 3’
cDNA
TTTTTp-5’
3’-CCCCCCCC
5’-pGGGG-OH 3’
-3’
5’-pGGGG 3’
cDNA
TTTTTp-5’
3’-CCCCCCCC
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences

13. Treatment of cDNA ends
Solution: 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’-pGGGG
TTTTTp-5’
3’-CCCCCCCC 1. 3’
-3’
5’-pGGGG
TTTTTp-5’
3’-CCC 2. 3’
-3’
5’-pGGGG
TTTTTp-5’
3’-CCCC 3.
HO-CCCAATTCGGGGGG
3’-GGCTTAAGCCCCCC 3’
-3’
5’-pGGGG
TTTTTp-5’
3’-CCCC
CCGAATTCGG-3’
GGCTTAAGCC-OH 4. 3’
-3’
5’-pGGGG
TTTTTp-5’
3’-CCCC
5’-pAATTCGGGGGG
3’-GCCCCCC
CCG-3’
GGCTTAAp-5’
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences

14. 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 S P
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences

15. I 3 Screening Procedure Screening for libraries
Colony and plaque hybridization
Expression screening
Hybrid arrest and release
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences

16. 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.
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences

17. 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-labeled
DNA complementary to
Gene of interest
Expose to film
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences

18. 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.
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences

19. Colony and plaque hybridization with probe-III
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.
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.
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences

20. Expression screening-I
Definition: 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.
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences

21. 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.
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences

22. Hybrid arrest
mRNA
Hybrid arrested translation: cDNAhybridized 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.
cDNA
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences

23. Hybrid release
cDNA
Hybrid release translation: cDNAhybridized mRNAproteins
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
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences

24. That’s all for Section I
Section I: Gene libraries and screening
Yang Xu, College of Life Sciences