dna libraries

8/6/2019 DNA Libraries

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DNA LibrariesDNA Libraries



DNA librariesDNA libraries -- a collections of DNAa collections of DNA

sequencessequences• DNA libraries, like conventional libraries, are

used to collect and store information.• In DNA libraries, the information is stored as aset of DNA molecules.

• All DNA libraries are collections of DNAfragments that represent a particular biologicalsystem of interest.

• The two most common uses for these DNAcollections are DNA sequencing and genecloning.



DNA librariesDNA libraries -- a collections of DNAa collections of DNA

sequencessequences• A DNA library is a collection of clones of

DNA designed so that there is a highprobability of finding any particular piece of the source DNA in the collection.

• DNA libraries can be made using highlyefficient cloning vectors such as lambdaphages, plasmids, cosmids, P1 phagesand bacterial or yeast artificialchromosomes.



Types of DNA LibrariesTypes of DNA Libraries

• The genomic librarygenomic library contains DNA

fragments representing the entire genomeof an organism.

• The cDNAcDNA librarylibrary contains onlycomplementary DNA moleculessynthesized from mRNA molecules in acell.



Genomic LibraryGenomic Library

• Are made from total nuclear DNA of an

organism or species.

• DNA is cut into clonable size pieces asrandomly as possible using restrictionendonuclease



Genomic LibraryGenomic Library

• Genomic libraries contain whole genomicfragments including gene exons and introns,gene promoters, intragenic DNA, centromericDNA, origins of replication, etc

exon exon exonpromoter intragenic DNA

DNA fragments

vector GenomicDNALibrary



Construction of DNA LibraryConstruction of DNA Library

• The library is made by inserting these millions of fragmentsof DNA en masse into λ bacteriophage plasmids.

• This allows the genes to be grown up (cloned) in E. coli .• The library can be screened for DNA fragments or

particular genes.



Screening aScreening a LamdaLamda Phage LibraryPhage Library

• Recombinant phage particles are plated onto

E. coli cells resulting in plaque formation• Phage particles and extracellular viral genomicDNA are transferred to a nitrocellulose filter

• The nitrocellulose filter is incubated with aradiolabeled probe for the desired gene• Autoradiograph will locate the clone with the

desired gene• This clone can be isolated and inoculated into

new host cells for further amplification



cDNAcDNA

LibraryLibrary

• The advantage of cDNA library is that it contains

only the coding region of a genome.exon exon exonpromoter intragenic DNA

Reverse transcriptase

cDNA

vector cDNA Library

transcription

polyA tailprocessing

Oligo dT primer



Genomic andGenomic and cDNAcDNA LibrariesLibraries

vector GenomicDNA Library

exon exon exon promoter intragenic DNA

cDNA

vector cDNA Library



Vectors for DNA LibrariesVectors for DNA Libraries

• Genomic libraries – λ -phage - 9-23 kb → convenient and easy to handle – Cosmids - 30-45

– PAC, BAC, YAC →artificial chromosomes,accommodate large fragments

• cDNA libraries-λ -phage - 9-23 kb → provides selection for longer

cDNAs

-conventional plasmids → high level of expression of proteins



Life Cycle of aLife Cycle of a BacteriophageBacteriophage

((BacteriophageBacteriophage Lambda,Lambda, λλ ))



Construction and Screening a LambdaConstruction and Screening a Lambda

Phage LibraryPhage Library



Construction of DNA LibraryConstruction of DNA Library

Endonuclease

digestion

Ligate with T4 DNA Ligase

Plate onE. coli



TheThe

BacteriophageBacteriophage

GenomeGenome

Replaceable region Lytic regionTailHead

Simplified map of the λ phage genome

Region of the λgenome can bereplaced byexogenous DNA



mRNAmRNA →→ cDNAcDNAAAAAn

5’ 3’

AAAAn5’ 3’

d TTTT 15

mRNA

mRNA

AAAAn

5’ 3’

d TTTT 15

mRNA

cDNA

d TTTT 15cDNA

Primer annealing

Reverse transcriptase

Alkali

cDNA

DNA polymerase



Screening DNA LibrariesScreening DNA Libraries

• Probe hybridization screens – Using antibody probes to detect antigenic fusion proteins -

Protein-specific antibodies can be used as "probes" toimmunologically detect bacterial fusion proteins encoding theprotein antigen. Vectors such as λgt11 and lambda Zap phagecan be used.

• Functional screens – Screening cDNA libraries based on differential expression -

In this screening strategy, mRNA is isolated from cells thatexpress the phenotype (or protein) of interest (+), and from cellsthat do not (-). The (+)mRNA is converted to radioactive (+)cDNAusing RTase and 32 P-dNTPs, and then hybridized to a massexcess of (-)mRNA using solution hybrdization. By removing thedouble-stranded cDNA:mRNA heteroduplexes, it is possible toobtain an enriched probe containing (+)cDNA sequences.



Blotting and HybridizationBlotting and Hybridization

1. A mixture of nucleicacid are separated byelectrophoresis.

2. Later the nucleic acidare transferred fromthe gel to a membraneby capillary action.

3. Specific bands aredetected by

hybridization.



DNA Blot: Southern BlotDNA Blot: Southern Blot



RNA blotRNA blot :: NorthenNorthen BlotBlot•• It can be used to determine the temporal and spatial locationsIt can be used to determine the temporal and spatial locations of of RNA expression byRNA expression by ““runningrunning ”” an RNA blot, often referred to as aan RNA blot, often referred to as anorthern blot.northern blot.


http://slidepdf.com/reader/full/dna-libraries 21/37Sequence-based process for detecting a particular gene - use of probes

CloningCloning -- specific DNA detection byspecific DNA detection by

hybridizationhybridization



PCR Based Screening DNA LibraryPCR Based Screening DNA Library

A B C D E F G H I

1234

5678



Design of probeDesign of probe

CloningCloning -- specific DNA detection byspecific DNA detection by

hybridizationhybridization



• cDNA library made more specialized by fusing a reporter gene to cDNAsequence• GFP (green fluorescent protein)fused to allow study of location andmovement of protein

From genes to genomesFrom genes to genomes -- creation of creation of DNA librariesDNA libraries



Regulation of Gene Expression:Regulation of Gene Expression:

LacLac OperonOperon

lacZ lacY lacA

Inducible genesrequired duringlactose metabolism

RNA polymerase

lacZ lacY lacA

No transcriptionRepressor

IPTG



http://mgl.scripps.edu/people/goodshttp://mgl.scripps.edu/people/goods

ell/pdb/pdb39/lacell/pdb/pdb39/lac --operon.gif operon.gif



BetaBeta --galactosidasegalactosidase



• X-gal (chromogenic substrate that yields blue

product when cleaved by β -galactosidase) andthe white colonies (non-functional B-galactosidase) are the ones with plasmid +

insert; the blue ones have plain plasmid.



Blue/White ScreeningBlue/White Screening

lacZlacZMCS

β -Galactosidase gene in frame with MCS

Transcription of lacZ occurs and β -galactosidase protein degrades X gal,thus blue colonies are formed.

lacZ lacZ

β -Galactosidase gene is out of frame with MCS

Transcription of lacZ does not occur and defective β -galactosidase results,thus white colonies are formed.

InsertedDNA

MCS

Plasmid DNA

Plasmid DNA



LB agar plate showing the result of LB agar plate showing the result of

a blue white screen.a blue white screen.



Site Directed MutagenesisSite Directed Mutagenesis

The Nobel Prize inChemistry 1993Michael Smith

Using siteUsing site --directed mutagenesis the information in thedirected mutagenesis the information in thegenetic material can be changed. This allows thegenetic material can be changed. This allows theinvestigation of the gene and/or protein biological role.investigation of the gene and/or protein biological role.Researchers can study in detail how proteins functionResearchers can study in detail how proteins functionand how they interact with other biological molecules.and how they interact with other biological molecules.



Site Directed MutagenesisSite Directed Mutagenesis

http://www.biochemj.org/bj/377/0171/bj3770171.htm

Effects of site-directed mutagenesis on PP1inhibition by wild-type and mutant GST-GBPIs

(Two Glutamic acid for Lysine)

(Tryptophan for Alanine)

(Tyrosine for Lysine)



Home Work:Home Work:

Nobel Prize in Medicine 2007Nobel Prize in Medicine 20073 Win Nobel in Medicine for Gene Manipulation

Left, Dan Sears/European Pressphoto Agency; Andrew Weltch/European Pressphoto Agency; Dirk Douglass/Reuters

From left, Oliver Smithies, Martin J. Evans and Mario R. Capecchi.



LaboratorioLaboratorio

15151.51.5221.51.510102 (Experimental)2 (Experimental)

15153.53.5------1.51.510101 (Control)1 (Control)

VolumenVolumenFinalFinal

µµll HH 22OOµµll EcoRIEcoRI(1/10)(1/10)

µµll BufferBuffer10X10X

DNADNA µµllminiprepminiprep

TuboTubo

POSIBLES PLASMIDOSDESCONOCIDOS: pGLO, pGEM, pUC19



ReferencesReferences

• http://www.ncbi.nlm.nih.gov/books/bv.fcgi?index

ed=google&rid=mcb.section.1611• Liu Qing-Rong, et al. 2004. GBPI, a novelgastrointestinal- and brain-specific PP1-

inhibitory protein, is activated by PKC andinactivated by PKA. 2004. Biochem. J. 377:171–181.See http://www.biochemj.org/bj/377/0171/bj3770171.htm

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