1. Section I – Gene libraries and screening

2. Contents I1 Genomic libraries I2 cDNA 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

3. 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
(made from genomic DNA)
Gene library
cDNA libraries
(made from cDNA- copy of mRNA)

4. 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

5. 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

6. 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.

7. I1 Genomic libraries — Genomic DNA
Eukaryotes
Purify genomic DNA
Correct size for cloning into the chosen vector:
Physical shearing and restriction enzyme digestion
Prokaryotes
Clone the fragments into vectors

8. 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

9. 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.

10. (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’

11. 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).

12. I1 Genomic libraries — Vectors
According to genome’s size, select a proper vector to construct a library .
Vectors Plasmid phageλ cosmid YAC
insert (kb)

13. 2.Ligation λ replacement vector Library constructed
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

14. 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

15. 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

16. 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

17. Most eukaryotic mRNAs are polyadenylated at their 3’ ends.
oligo (dT) can be bound to the poly(A) tail and used to recover the mRNA.
3’-AAAAAAAAAAn
5’- cap

18. Three methods:
Traditional method was done by pass a preparation of total RNA down a column of oligo (dT)-cellulose.
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.
Lying cells and then preparing mRNA-ribosome complexes on sucrose gradients.

19. 100mM NaCl
rRNA and tRNA
10mM Tris, 1mM EDTA, poly(A) and -oligo(dT)

20. 3. Check the mRNA integrity
Make sure that the mRNA is not degraded.
Methods:
Translating the mRNA : use cell-free translation system as wheat germ extract or rabbit reticulocyte lysate to see if the mRNAs can be translated
Analysis the mRNAs by gel electrophoresis: Use agarose or polyacrylamide gels

21. 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)

22. I2 cDNA libraries — Synthesis of cDNA
The first strand and Second strand synthesis

23. I2 cDNA libraries — Treatment of cDNA
HO-CCGAATTCGGGGGG
DNA linker:
3’-GGCTTAAGCCCCCC
HO - CGGGGGG
DNA adaptor:
3’-TTAAGCCCCCC
Blunt end ligation of large fragments is not efficient, so we have to use special linkers to create sticky ends for cloning.

25. 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 )

26. 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.

27. 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

28. 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.

29. 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

30. 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 (radio-
actively labeled )
Wash to remove unhybri-
dized probe and visualize
Antibody or enzyme
(modified nucleotide labeled)
Line up the hybridizated region or
repeated hybridization

31. 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

33. 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).

34. 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

35. I3 Screening procedures — Chromosome walking
Definition: To clone the desired gene by repeated isolating adjacent genomic clones from the library.

37. 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.

38. 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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