DNA Libraries

DNA libraries - a collections of DNA

sequences
• DNA libraries, like conventional libraries, are
used to collect and store information.
• In DNA libraries, the information is stored as a
set of DNA molecules.
• All DNA libraries are collections of DNA
fragments that represent a particular biological
system of interest.
• The two most common uses for these DNA
collections are DNA sequencing and gene
cloning.
DNA libraries - a collections of DNA
sequences
• A DNA library is a collection of clones of
DNA designed so that there is a high
probability of finding any particular piece of
the source DNA in the collection.
• DNA libraries can be made using highly
efficient cloning vectors such as lambda
phages, plasmids, cosmids, P1 phages
and bacterial or yeast artificial
chromosomes.
 Types of DNA Libraries
• The genomic library contains DNA
fragments representing the entire genome
of an organism.
• The cDNA library contains only
complementary DNA molecules
synthesized from mRNA molecules in a
cell.
 Genomic Library
• Are made from total nuclear DNA of an
organism or species.

• DNA is cut into clonable size pieces as

randomly as possible using restriction
endonuclease
 Genomic Library
• Genomic libraries contain whole genomic
fragments including gene exons and introns,
gene promoters, intragenic DNA, centromeric
DNA, origins of replication, etc

Genomic
vector
DNA
Library

DNA fragments

promoter exon exon exon intragenic DNA

 Construction of DNA Library
• The library is made by inserting these millions of fragments
of 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 a Lamda Phage Library
• Recombinant phage particles are plated onto
E. coli cells resulting in plaque formation
• Phage particles and extracellular viral genomic
DNA are transferred to a nitrocellulose filter
• The nitrocellulose filter is incubated with a
radiolabeled 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
 cDNA Library
• The advantage of cDNA library is that it contains
only the coding region of a genome.

promoter exon exon exon intragenic DNA

transcription

processing
polyA tail

Reverse transcriptase Oligo dT primer

cDNA

cDNA Library
vector
Genomic and cDNA Libraries
Genomic
vector
DNA Library

promoter exon exon exon intragenic DNA

cDNA

cDNA Library
vector
 Vectors 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 a Bacteriophage
(Bacteriophage Lambda, λ)
Construction and Screening a Lambda
Phage Library
Construction of DNA Library
Endonuclease
digestion

Ligate with T4 DNA Ligase

Plate on
E. coli
The Bacteriophage Genome

Simplified map of the λ phage genome

Head Tail Replaceable region Lytic region

Region of the λ
genome can be
replaced by
exogenous DNA
 mRNA → cDNA
5’ 3’
mRNA AAAAn

Primer annealing

mRNA 5’ 3’
AAAAn
d TTTT15

Reverse transcriptase

mRNA 5’ 3’
AAAAn
cDNA d TTTT15

Alkali
cDNA d TTTT15

DNA polymerase

cDNA
 Screening DNA Libraries
• Probe hybridization screens
– Using antibody probes to detect antigenic fusion proteins -
Protein-specific antibodies can be used as "probes" to
immunologically detect bacterial fusion proteins encoding the
protein antigen. Vectors such as λgt11 and lambda Zap phage
can be used.
• Functional screens
– Screening cDNA libraries based on differential expression -
In this screening strategy, mRNA is isolated from cells that
express the phenotype (or protein) of interest (+), and from cells
that do not (-). The (+)mRNA is converted to radioactive (+)cDNA
using RTase and 32P-dNTPs, and then hybridized to a mass
excess of (-)mRNA using solution hybrdization. By removing the
double-stranded cDNA:mRNA heteroduplexes, it is possible to
obtain an enriched probe containing (+)cDNA sequences.
 Blotting and Hybridization

1. A mixture of nucleic
acid are separated by
electrophoresis.
2. Later the nucleic acid
are transferred from
the gel to a membrane
by capillary action.
3. Specific bands are
detected by
hybridization.
DNA Blot: Southern Blot
 RNA blot : Northen Blot
• It can be used to determine the temporal and spatial locations of
RNA expression by “running” an RNA blot, often referred to as a
northern blot.
Cloning - specific DNA detection by
hybridization

Sequence-based process for detecting a particular gene - use of probes

PCR Based Screening DNA Library
1
2
3
4
5
6
7
8

ABCDEFGHI
Cloning - specific DNA detection by
hybridization

Design
Design of
of probe
probe
 From genes to genomes - creation of
DNA libraries

• cDNA library made more specialized by fusing a reporter gene to cDNA

sequence
• GFP (green fluorescent protein)fused to allow study of location and
movement of protein
Regulation of Gene Expression
• Mechanisms in prokaryotes
– Transcriptional
• Operons
– Translational
• Mechnisms in Eukaryotes
– Transcriptional
– Posttranscriptional
– Translational
– Posttranslational
Regulation of Gene Expression:
Lac Operon
RNA polymerase
Inducible genes
required during
lactose metabolism

lacZ lacY lacA

IPTG

lacZ lacY lacA

/

No transcription
Repressor
In the lab IPTG induces gene
expression of lac system
lacZ lacY lacA

No transcription

IPTG
(Isopropyl-[beta]-D-thiogalactopyranoside )
β-galactosidase

β-galactosidase acts on Xgal

(a blue chromogenic
substrate), thus colonies
containing plasmid with inserts
in its MCS, will turn white.
http://mgl.scripps.edu/people/goods
ell/pdb/pdb39/lac-operon.gif
Beta-galactosidase
• X-gal (chromogenic substrate that yields blue
product when cleaved by β-galactosidase) and
the white colonies (non-functional B-
galactosidase) are the ones with plasmid +
insert; the blue ones have plain plasmid.
 Blue/White Screening
β-Galactosidase gene in frame with MCS

Plasmid DNA
lacZ MCS lacZ

Transcription of lacZ occurs and β-galactosidase protein degrades X gal,

thus blue colonies are formed.

β-Galactosidase gene is out of frame with MCS

Plasmid DNA
lacZ Inserted MCS lacZ
DNA

Transcription of lacZ does not occur and defective β-galactosidase results,

thus white colonies are formed.
LB agar plate showing the result of
a blue white screen.
 Site Directed Mutagenesis
Using site-directed mutagenesis the information in the
genetic material can be changed. This allows the
investigation of the gene and/or protein biological role.
Researchers can study in detail how proteins function
and how they interact with other biological molecules.

The Nobel Prize in

Chemistry 1993
Michael Smith
Site Directed Mutagenesis
Effects of site-directed mutagenesis on PP1
inhibition by wild-type and mutant GST-GBPIs

(Two Glutamic acid for Lysine)

(Tryptophan for Alanine)

(Tyrosine for Lysine)

http://www.biochemj.org/bj/377/0171/bj3770171.htm
 Home Work:
Nobel Prize in Medicine 2007
3 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.

 Laboratorio
Tubo DNA µl µl Buffer µl EcoRI µl H2O Volumen
miniprep 10X (1/10) Final

1 (Control) 10 1.5 --- 3.5 15

2 (Experimental) 10 1.5 2 1.5 15

POSIBLES PLASMIDOS
DESCONOCIDOS: pGLO, pGEM, pUC19
 References
• http://www.ncbi.nlm.nih.gov/books/bv.fcgi?index
ed=google&rid=mcb.section.1611
• Liu Qing-Rong, et al. 2004. GBPI, a novel
gastrointestinal- and brain-specific PP1-
inhibitory protein, is activated by PKC and
inactivated by PKA. 2004. Biochem. J. 377:
171–181.
See http://www.biochemj.org/bj/377/0171/bj3770171.htm