Gene libraries and screening

Genomic libraries
cDNA libraries
Screening procedures
Introduction
• The use of genetic information is a
powerful tool that today is becoming more
readily available to scientis...
I1 Genomic libraries

Gene library: a collection of different DNA
sequence from an organism, each of which
has been cloned...
I1 Genomic libraries

Size of library (ensure enough clones)

must contain a certain number of
recombinants for there to b...
I1 Genomic libraries
For example :for a probability of 0.99 with
insert sizes of 20 kb these values for the E.coli
(4.6×10...
I1 Genomic libraries

Genomic DNA libraries
eukaryotes
Purify genomic DNA
prokaryotes
Fragment this DNA : physical shearin...
I1 Genomic libraries
To make a representative genomic libraries ,
genomic DNA must be purified and then
broken randomly in...
I1 Genomic libraries

Break DNA into fragments randomly:

Physical shearing :
pipeting, mixing

Restriction enzyme digesti...
I1 Genomic libraries

Selection of restriction enzyme
1. Ends produced (sticky or blunt) &
The cleaved ends of the vector ...
Generating A Genomic Library
•

•

•
•

λ-phage is treated with restriction
enzymes that produce λ arms with
sticky end. T...
λ-phage as a Vector
•

The genomic library is
generated by using λ-phage for
the following reasons.

1.

A large number of...
λ-phage as a Vector (Cont.)
•

•

•

An infected E.Coli will produce
what are know as concatomers
(which is the viral geno...
Packaging of the Recombinant DNA
•
•
•
•
•
•
•

To prepare the phage an E.coli cell is infected with a mutant λ-phage
that...
I1 Genomic libraries

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

Vectors

...
λ phage vector in cloning
Long (left)
arm
cos

short (right)
arm

Exogenous DNA
(~20-23 kb)

Long (left)
arm

cos

short (...
0.preparation of
arm and genomic
inserts

1. Ligation

λ replacement
vector cloning
2. Packing with a
mixture of the phage...
Gene libraries and screening

I

cDNA libraries

mRNA isolation, purification
Check theRNA integrity
Fractionate and enric...
I 2 cDNA libraries

cDNA libraries

1.No cDNA library was made
from prokaryotic mRNA.
• Prokaryotic mRNA is very unstable
...
I 2 cDNA libraries

cDNA libraries
2.cDNA libraries are very useful
for eukaryotic gene analysis
• Condensed protein encod...
I 2 cDNA libraries

mRNA isolation
• Most eukaryotic mRNAs are polyadenylated at
their 3’ ends
5’ cap

AAAAAAAAAAn

• olig...
I 2 cDNA libraries
I2 cDNA libraries

Three methods to isolate mRNA.
1.Traditionally method was done by pass a
preparation of total RNA down ...
I2 cDNA libraries

Check the mRNA integrity
Make sure that the mRNA is not
degraded. Methods:
Translating the mRNA : use c...
I2 cDNA libraries

Cloning the particular mRNAs
Is useful especially one is trying to clone a
particular gene rather to ma...
I2 cDNA libraries

Synthesis of cDNA :
First stand synthesis: materials as
reverse transcriptase ,primer( oligo(dT) or
hex...
I2 cDNA libraries
5’

5’
3’
5’
3’-CCCCCCC
5’-pGGGG-OH
3’-CCCCCCC

5’-pGGGG
3’-CCCCCCC

mRNA
Reverse transcriptase
Four dNT...
Duplex cDNA

5’-pGGGG
3’-CCCCCCC

-3’
TTTTTp-5’

Single strand-specific nuclease
5’-pGGGG
3’-CCC

-3’
TTTTTp-5’

Klenow po...
I2 cDNA libraries

Treatment of cDNA ends

Blunt and ligation of large fragment is not
efficient, so we have to use specia...
I2 cDNA libraries

Ligation to vector
Any vectors with an E.coRI site would suitable
for cloning the cDNA.

The process :
...
Gene libraries and screening

Screening procedures
Screening
Colony and plaque hybridization
Expression screening
Hybrid a...
I3 Screening procedures

Screening
The process of identifying one particular
clone containing the gene of interest from
am...
I3 Screening procedures

Screening libraries

Searching the genes of interest in a DNA library

Hybridization to identify ...
I3 Screening procedures

Colony and plaque hybridization
Transfer the DNA in the plaque or colony to a
Nylon or nitrocellu...
I3 Screening procedures

Expression screening
Identify the protein product of an
interested gene

1. Protein activity
2. W...
I3 Screening procedures

Expression screening
If the inserts are cloned into an expression
sites, it may be expressed. The...
I3 Screening procedures

Expression screening
Antibodies can be used to screen the
expression library.

The procedure

‘Pl...
I3 Screening procedures

Hybrid arrest and screen
Individual cDNA clones or pools of clones can
be used to hybridize to mR...
I3 Screening procedures

I3-5 Chromosome walking
Definition: To clone the desired gene by
repeated isolating adjacent
geno...
I3 Screening procedures

Process:

1. Prepare a probe from the end insert .
2.The probe are used to re-screen the library
...
Vector arm

}

}

Restriction

Genomic clone insert

Vector arm

Prepare probe from
ends of insert

Re-screen genomic
libr...
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Dna library lecture-Gene libraries and screening

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  • Dna library lecture-Gene libraries and screening

    1. 1. Gene libraries and screening Genomic libraries cDNA libraries Screening procedures
    2. 2. Introduction • The use of genetic information is a powerful tool that today is becoming more readily available to scientists. • In order to use this powerful tool it necessary to know how to navigate throughout the entire genome. The human genome is about 3 x 10E9 bp. • In humans this project is known as Human Genome Project.
    3. 3. I1 Genomic libraries Gene 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)
    4. 4. I1 Genomic libraries Size of library (ensure enough clones) must contain a certain number of recombinants for there to be a high probability of it containing any particular sequence The formula to calculate the number of recombinants: ln (1-P) N= ln (1-f) P: desired probability f : the fraction of the genome in one insert
    5. 5. I1 Genomic libraries For example :for a probability of 0.99 with insert sizes of 20 kb these values for the E.coli (4.6×106 bp) and human (3×109 bp) genomes are : N E.coli= Nhuman= ln( 1-0.99) ln[1-(2×104/4.6×106)] ln(1-0.99) = 1.1 ×103 = 6.9 ×105 ln[1-(2 ×104/3 ×109)] These values explain why it is possible 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.
    6. 6. I1 Genomic libraries Genomic DNA libraries eukaryotes Purify genomic DNA prokaryotes Fragment this DNA : physical shearing and restriction enzyme digestion Clone the fragments into vectors
    7. 7. I1 Genomic libraries To make a representative genomic libraries , genomic DNA must be purified and then broken randomly into fragments that are correct in size for cloning into the chosen vector. Purification of genomic DNA : Eukaryotes :prepare cell nuclei remove protein, lipids and other unwanted macromolecules by protease digestion and phase extraction. Prokaryotes :extracted DNA directly from cells
    8. 8. I1 Genomic libraries Break DNA into fragments randomly: Physical shearing : pipeting, mixing Restriction enzyme digestion: partial digestion is preferred to get a greater lengths of DNA fragments.
    9. 9. I1 Genomic libraries Selection of restriction enzyme 1. Ends produced (sticky or blunt) & The cleaved ends of the vector to be cloned Sau3A: 5’-/GATC-3’, less selectivity BamH1: 5’-G/GATCC 2. Whether the enzyme is inhibited by DNA modifications (CpG methylation in mammals 3. Time of digestion and ratio of restriction enzyme to DNA is dependent on the desired insert size range.
    10. 10. Generating A Genomic Library • • • • λ-phage is treated with restriction enzymes that produce λ arms with sticky end. These arms contain all the lytic genetic information that is needed for replication and produces room for insertion of new genetic information. DNA sequence is obtain from the cell of interest. It is cleaved with restriction enzymes that produce 20kb fragments that have complementary sticky ends. Both are mixed in equal amounts and are treated with a DNA ligase that cleaves them together. Afterward the entire combined sequence is packed to the phage head.
    11. 11. λ-phage as a Vector • The genomic library is generated by using λ-phage for the following reasons. 1. A large number of λ phage can be screened simultaneously (5 x 10E4 phage plagues). λ phage as a higher transformation efficiency about 1000 times higher compared to a plasmid. 2. • • The vector as to maintain its lytic growth. Lysogenic pathway and other viral genes that are not important are replaced with the DNA to be cloned.
    12. 12. λ-phage as a Vector (Cont.) • • • An infected E.Coli will produce what are know as concatomers (which is the viral genome) on either site of the concatomers there is a site called COS Site. Two proteins recognize this site A protein and Nu protein, which will lead to the insertion of the λ DNA into the phage head. The chromosomal DNA that lacks the COS sites will not enter the phage head. Once the genetic information is inserted the tail will assemble. A 50kb can be inserted into the phage.
    13. 13. Packaging of the Recombinant DNA • • • • • • • To prepare the phage an E.coli cell is infected with a mutant λ-phage that as a defective “A-protein” (which is one of two genes that are responsible for packaging genetic information). Therefore the E.Coli accumulates empty heads and also preassembled tails. Once enough heads and tails are assembled we lysate the E.Coli cells. To the mixture of heads and tail we add isolated A protein (obtained from E.Coli infected with λ-phage). In the next step we add the recombinant DNA that has the λ-phage genetic information (which also includes COS sites). At this point we have a mixture containing mutant λ-phage heads and tails. There is isolated A protein and recombinant DNA containing λphage genetic information with COS sites. Therefore we have all the components necessary to package the recombinant DNA into the λ-phage head. Once the information is inserted the tail assembles and we have an infectious phage that contains the recombinant DNA sequence.
    14. 14. I1 Genomic libraries Vectors According to genome’s size,we can select a proper vector to construct a library . Vectors Plasmid insert (kb) 5 phageλ 23 cosmid 45 YAC 1000 The most commonly chosen genomic cloning vectors are λ relacement 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 digested
    15. 15. λ phage vector in cloning Long (left) arm cos short (right) arm Exogenous DNA (~20-23 kb) Long (left) arm cos short (right) arm cos cos Exogenous DNA (~20-23 kb)
    16. 16. 0.preparation of arm and genomic inserts 1. Ligation λ replacement vector cloning 2. Packing with a mixture of the phage coat proteins and phage DNAprocessing enzymes 3. Infection and formation of plaques Library constructed
    17. 17. Gene libraries and screening I cDNA libraries mRNA isolation, purification Check theRNA integrity Fractionate and enrich mRNA Synthesis of cDNA Treatment of cDNA ends Ligation to vector
    18. 18. I 2 cDNA libraries cDNA libraries 1.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.
    19. 19. I 2 cDNA libraries cDNA libraries 2.cDNA libraries are very useful for eukaryotic gene analysis • Condensed protein encoded gene libraries, have much less junk sequences. • cDNAs have no introns → genes can be expressed in E. coli directly • Are very useful to identify new genes • Tissue or cell type specific (differential expression of genes)
    20. 20. I 2 cDNA libraries mRNA isolation • Most eukaryotic mRNAs are polyadenylated at their 3’ ends 5’ cap AAAAAAAAAAn • oligo (dT) can be bound to the poly(A) tail and used to recover the mRNA.
    21. 21. I 2 cDNA libraries
    22. 22. I2 cDNA libraries Three methods to isolate mRNA. 1.Traditionally 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.Alternative route of isolating mRNA is lysing cells and then preparing mRNAribosome complexes on sucrose gradients
    23. 23. I2 cDNA libraries 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 elctrophoresis: use agarose or polyacrylamide gels
    24. 24. I2 cDNA libraries 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: clone the hormone induced mRNAs (substrated cDNA library)
    25. 25. I2 cDNA libraries Synthesis of cDNA : First stand synthesis: materials as reverse transcriptase ,primer( oligo(dT) or hexanucleotides) and dNTPs Second strand synthesis: best way of making full-length cDNA is to ‘tail’ the 3’end of the first strand and then use a complementary primer to make the second.
    26. 26. I2 cDNA libraries 5’ 5’ 3’ 5’ 3’-CCCCCCC 5’-pGGGG-OH 3’-CCCCCCC 5’-pGGGG 3’-CCCCCCC mRNA Reverse transcriptase Four dNTPs mRNA AAAAA-3’ HO-TTTTTP-5’ cDNA AAAAA-3’ TTTTTP-5’ cDNA AAAAA-3’ TTTTTP-5’ Terminal transferase dCTP mRNA Alkali (hydrolyaes RNA) Purify DNA oligo(dG) TTTTTP-5’ Klenow polymerase or reverse Transcriotase Four dNTPs -3’ TTTTTP-5’ Duplex cDNA cDNA The first strand synthesis
    27. 27. Duplex cDNA 5’-pGGGG 3’-CCCCCCC -3’ TTTTTp-5’ Single strand-specific nuclease 5’-pGGGG 3’-CCC -3’ TTTTTp-5’ Klenow polymerase treat with E.coRI methylase 5’-pGGGG 3’-CCCC Add E.colRI linkers using T4 DNA ligase HO-CCGAATTCGGGGGG 3’-GGCTTAAGCCCCCC -3’ TTTTTp-5’ HO-CCG/AATTCGG-3’ 3’-GGCTTAA/GCC-OH CCGAATTCGG-3’ TTTTTGGCTTAAGCC-OH E.colRI digestion 5’-pAATTCGGGGGG 3’-CCCCCCC CCG-3’ TTTTTGGCTTAAp-5’ Ligate to vector and transfom Second strand synthesis
    28. 28. I2 cDNA libraries Treatment of cDNA ends Blunt and ligation of large fragment is not efficient, so we have to use special acid 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) Tailing with terminal transferase or using adaptor molecules Restriction enzyme digestion (E.coRI )
    29. 29. I2 cDNA libraries Ligation to vector Any vectors with an E.coRI site would suitable for cloning the cDNA. The process : Dephosphorylate the vector with alkaline phosphatase Ligate vector and cDNA with T4 DNA ligase (plasmid or λ phage vector)
    30. 30. Gene libraries and screening Screening procedures Screening Colony and plaque hybridization Expression screening Hybrid arrest and release Chromosome walking (repeat screening)
    31. 31. I3 Screening procedures 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.
    32. 32. I3 Screening procedures Screening libraries Searching the genes of interest in a DNA library Hybridization to identify the interested DNA or its RNA product 1. Radiolabeled probes which is complementary to a region of the interested gene Probes: • An oligonucleotide derived from the sequence of a protein product of the gene • A DNA fragment/oligo from a related gene of another species 2. Blotting the DNA or RNA on a membrane 3. Hybridize the labeled probe with DNA membrane (Southern) or RNA (Northern) membrane
    33. 33. 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. Hybridization (in a solution (Alkali treatment) Containing Nucleic acid probe) X-ray film(radioactively labeled ) Wash to remove unhybridization probe and visualize Line up the hybridizated region or repeated hybridization antibody or enzyme (modified nucleotide labeled
    34. 34. I3 Screening procedures Expression screening Identify the protein product of an interested gene 1. Protein activity 2. Western blotting using a specific antibody
    35. 35. 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. However, this screening may miss the right clone
    36. 36. I3 Screening procedures Expression screening Antibodies can be used to screen the expression library. The procedure ‘Plaque lift’ ( taken by placing a membrane on the dish of plaque) Immersed in a solution of the antibody Detected by other antibodies Repeat cycles of screening to isolate pure plaques
    37. 37. I3 Screening procedures Hybrid arrest and screen Individual cDNA clones or pools of clones can be used to hybridize to mRNA preparation Hybrid arrest :translate the mRNA population directly, and the inhibition of translation of some products detected. Hybrid release translation : purify the hybrids and the hybridized mRNAs released from them and translated, it identifies the protein encoded by the cDNA clone
    38. 38. I3 Screening procedures I3-5 Chromosome walking Definition: To clone the desired gene by repeated isolating adjacent genomic clones from the library. to obtain overlapping genomic clones that represent progressively longer parts of a particular chromosome .
    39. 39. I3 Screening procedures Process: 1. Prepare a probe from the end insert . 2.The probe are used to re-screen the library by colony or plaque hybridization 3.Analyzed the new isolate clones and posited them relative to the starting clone. some will be overlapping. 4. Repeated the whole process using a probe from the distal end of the second clone.
    40. 40. Vector arm } } Restriction Genomic clone insert Vector arm Prepare probe from ends of insert Re-screen genomic library Restriction map new genomic clones } } Prepare new probes from distal ends of least overlapping insert. Re-screen genomic library . Restriction map new genomic clones Chromosome walking
    41. 41. Thanks
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