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cDNA Library. MB206. Making a DNA library . Types of Libraries. (3’UTR). (5’UTR). Genomic DNA libraries. -Contains the whole genome of an organism. -A restriction-enzyme is used to cut the genome (the DNA) at numerous locations. . polyA. cDNA. Reverse transcribe (and more). polyA.

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cdna library

cDNA Library

MB206

Angelia 09

types of libraries
Types of Libraries

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(3’UTR)

(5’UTR)

genomic dna libraries
Genomic DNA libraries

-Contains the whole genome of an organism.

-A restriction-enzyme is used to cut the genome (the DNA) at numerous locations.

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genomic libraries

polyA

cDNA

Reverse transcribe

(and more)

polyA

polyA

Clone in vector

Digest

DNA fragments

Genomic Libraries

cDNA library

Genomic DNA

mRNA

Genomic DNA library

Genomic DNA

Angelia 09

cdna libraries

polyA

cDNA

Reverse transcribe

(and more)

polyA

polyA

Clone in vector

Digest

DNA fragments

cDNA Libraries

cDNA library

Genomic DNA

mRNA

Genomic DNA library

Genomic DNA

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cdna libraries1

“complementary” DNA

[mRNA it is used to create it]

cDNA Libraries
  • Purify mRNA
    • mRNA-> single stranded cDNA using reverse transcriptase.
    • Single stranded cDNA -> double stranded cDNA (DNA polymerase and other “cloning tricks”).
    • Linkers added to cDNA & clone into vectors as seen in genomic DNA libraries

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slide9

Lodish, et al. 1999

Figure 7-14, 7-15

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genomic libraries1

polyA

Reverse transcribe

(and more)

cDNA

polyA

polyA

Clone in vector

Digest

DNA fragments

Genomic Libraries

cDNA library

Genomic DNA

mRNA

Genomic DNA library

Genomic DNA

Angelia 09

digest genomic dna with restriction enzymes
Digest genomic DNA with restriction enzymes
  • Which restriction enzyme should we select?

Consideration: Try not to cut the gene or operon of interest

    • V. fischeri LUX operon 9kb

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selecting a restriction enzyme
Selecting a restriction enzyme
  • What is the average size fragment that any given enzyme will yield?
    • If enzyme recognizes 6bp, statistically any given 6bp sequence will appear randomly every 46 bases (every 4096 bases)

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how often does the enzymes cut
How often does the enzymes cut?

A ‘four-base cutter’ recognition sequence would occur once every 44 = 256 bp

A ‘six-base cutter’ would give you fragments of about 4000 bp

An ‘eight-base cutter’ recognition sequence would occur once every 48 or 65,536 bp.

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how often do enzymes really cut
How often do enzymes really cut?
  • But also we need to consider G+C content of the genome:
    • V. fischeri G+C content = 40%
    • Sal I recognizes sites that are G+C rich (GTCGAC)  it will cut less often in Vibrio genomic DNA.

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slide16

Let’s stop

here and

think

about it!

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problem

Take your time!

PROBLEM:
  • The genomic DNA is 60% G+C
  • You want a 5000bp fragment
  • Which enzyme would you try first, Why?
    • SmaI (CCCGGG)
    • EcoRI (GAATTC)
    • MseI (TTAA)
    • SacI (GAGCTC)

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genomic libraries2

Digest

DNA fragments

Genomic Libraries

Genomic DNA library

Clone in vector

Genomic DNA

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choosing a vector
Choosing a Vector
  • Usually you select a vector (plasmid, λ, other) depending on how big you want your DNA fragments to be & the capacity of the vector.

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common vectors used in library construction
Common vectors used in library construction
  • Plasmids
    • Up to 10kb inserts
  • Modified Lambda phage
    • Up to 20kb inserts/40kbp for cosmids
  • Artificial cloning vectors
    • BAC- Vectors (bacterial artificial chromosome)

Up to 100-150kbp inserts

    • YAC-Vectors (yeast artificial chromosome)

Up to 500kbp inserts

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slide21

Lambda Library

Lodish, et al. Fig 7-12

Plasmid Library

Lodish, et al. Fig 7-1

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genomic libraries3

Digest

DNA fragments

Genomic Libraries

Genomic DNA library

Clone in vector

Genomic DNA

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slide25

cDNA libraries

  • 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.

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slide26

cDNA libraries

  • 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)

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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.
mRNA isolation

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slide28

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

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slide29

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)

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slide30

Synthesis of cDNA :

First stand synthesis:

materials as reverse transcriptase ,primer( oligo(dT) or hexanucleotides) and dNTPs (Fig 1.1)

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.

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slide31

mRNA

5’

AAAAA-3’

HO-TTTTTP-5’

Reverse transcriptase

Four dNTPs

mRNA

5’

AAAAA-3’

TTTTTP-5’

3’

cDNA

Terminal transferase

dCTP

mRNA

5’

AAAAA-3’

3’-CCCCCCC

TTTTTP-5’

cDNA

Alkali (hydrolyaes RNA)

Purify DNA oligo(dG)

5’-pGGGG-OH

3’-CCCCCCC

TTTTTP-5’

cDNA

Klenow polymerase or reverse

Transcriotase Four dNTPs

5’-pGGGG

-3’

3’-CCCCCCC

TTTTTP-5’

Duplex cDNA

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slide32

Duplex cDNA

5’-pGGGG

-3’

3’-CCCCCCC

TTTTTp-5’

Single strand-specific nuclease

5’-pGGGG

-3’

3’-CCC

TTTTTp-5’

Klenow polymerase

treat with E.coRI methylase

5’-pGGGG

-3’

3’-CCCC

TTTTTp-5’

Add E.colRI linkers

using T4 DNA ligase

HO-CCG/AATTCGG-3’

3’-GGCTTAA/GCC-OH

HO-CCGAATTCGGGGGG

CCGAATTCGG-3’

3’-GGCTTAAGCCCCCC

TTTTTGGCTTAAGCC-OH

E.colRI digestion

5’-pAATTCGGGGGG

CCG-3’

3’-CCCCCCC

TTTTTGGCTTAAp-5’

Ligate to vector and transfom

Fig2.1 Second strand synthesis

slide33

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 )

slide34

Ligation to vector

Any vectors with an EcoRI 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)

slide35

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 .

  • Using nucleic acid probe to screen the library based on hybridization with nucleic acids.
  • Analyze the protein product.

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slide36

Screening libraries

Searching the genes of interest in a DNA library

  • Hybridization to identify the interested DNA or its RNA product
  • 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
  • Blotting the DNA or RNA on a membrane
  • Hybridize the labeled probe with DNAmembrane (Southern) or RNA (Northern) membrane

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slide37

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

Containing Nucleic acid probe)

(Alkali treatment)

X-ray film(radio-

actively labeled )

antibody or enzyme

(modified nucleotide labeled

Wash to remove unhybri-

dization probe and visualize

Line up the hybridizated region or

repeated hybridization

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slide38

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

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