EXPRESSED SEQUENCE TAGS & NORTHERN BLOTTING

EXPRESSED SEQUENCE TAGSExpressed Sequence Tags (ESTs) are small pieces of DNA sequences that are usually 200-500 nucleotides long and are used to locate a gene within the genome.

Expressed sequence tags (ESTs) are fragments of mRNA sequences derived through single sequencing reactions performed on randomly selected clones from cDNA libraries.

WHAT ARE ESTScDNA libraries prepared from various organisms, tissues and cell lines using directional cloning.

Gridding of individual clones using robots.

For each clone, single-pass sequencing of both ends (5’ and/or 3’) of insert.

Deposit readable part of sequence in database

ESTs represent partial sequences of cDNA clones (300 bp -> 700 bp)

WHAT ARE ESTS

AAAA

mRNA

cDNA

Synthesis of 1 strand of DNA(Reverse Transcriptase)

RNA degradation Synthesis of 2 strand of DNA (DNA Polymerase)3’5’

3’

5’

Cloning &Sequencing

CDNA LIBRARIES

•Most are “native”, meaning that clone frequency reflects mRNA abundance

•Most are primed with oligo(dT), meaning that 3’ ends are heavily represented

•The complexity of libraries is extremely variable

•“Normalized” libraries are used to enrich for rare mRNAs

• Sequencing only the beginning portion of the cDNA produces 5' EST.

• 5' EST is obtained from the portion of a transcript that usually codes for a protein. These regions tend to be conserved across species and do not change much within a gene family . 

• Sequencing the ending portion of the cDNA molecule produces 3' EST . 

• 3' EST are likely to fall within non-coding, or untranslated regions (UTRs) , and therefore tend to exhibit less cross-species conservation than do coding sequences.

From cDNA to ESTs

EST databasesPublic EST databases• EMBL/GenBank have separate sections for EST sequences• ESTs are the most abundant entries in the databases (>60%)• ESTs are now separated by division in the databases:

-> human, mouse, plant, prokaryote, … (EMBL)• ESTs sequences are submitted in bulk, but do have to meet minimal quality criteria (“Phred” score >20%, ie <1% error)

Private EST databases(producing and selling access to EST data has proven to be a lucrative business…)• Human Genome Sciences (http://www.hgsi.com/) exploit the data itself, and getpatents on promising genes found in its databases

APPLICATION OF ETSIdentify unknow gene and to map their

positions within a genome.

Discovering new genes.

For obtaining data on gene expression and regulation.

Constructing genome mapping.

NORTHERN BLOTTING

NORTHERN BLOTTING• Northern blot analysis reveals information about RNA

identity, size, and abundance, allowing a deeper understanding of gene expression levels.

• With Northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation, morphogenesis, as well as abnormal or diseased conditions.

• Northern blotting involves the use of electrophoresis to separate RNA samples by size and detection with a hybridization probe complementary to part of or the entire target sequence.

• The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University.

• Northern blotting takes its name from its similarity to the first blotting technique, the Southern blot, named for biologist Edwin Southern.

• The major difference is that RNA, rather than DNA, is analyzed in the Northern blot.

PROCEDURE

• RNA is isolated from several biological samples.

• The RNA samples are separated according to their size on an agarose gel.

• The gel is then blotted on a nylon membrane or a nitrocellulose filter.

Sampleas are loaded on gel

The resulting gel following the run

• The membrane is placed in a dish containing hybridization buffer with a labeled probe (radioactively or chemically labeled). The probe is specifically designed for the sequence of interest. Thus, it will hybridize to the RNA on the blot that corresponds to the sequence of interest.

• The membrane is washed to remove unbound probe.

• The labeled probe is detected via autoradiography (if a radioactive probe is used) or via a chemiluminescence reaction (if a chemically labeled probe is used). In both cases this results in the formation of a dark band on an X-ray film.

• We can now compare expression patterns of the sequence of interest in the different samples.

The probe can be either ss-DNA or RNA.

Access probe is removed

APPLICATIONS

A standard for direct study of the gene expression at the level of mRNA .

Detection of mRNA transcript size .

Study of RNA splicing – can detect alternatively spliced transcripts .

Study RNA half life

DISADVANTAGES•  Risk of mRNA degradation during electrophoresis:

quality and quantification of expression are negatively affected.

•   High doses of radioactivity and formaldehyde are a risk for workers and the environment.

•   The sensitivity of northern blotting is relatively low in comparison with that of RT-PCR.

•   Detection with multiple probes is difficult.•   Use of ethidium bromide, DEPC and UV light

needs special training and attention.

ADVANTAGES

• The strength of this method is its simplicity.

• Specificity is relatively high.

• Sequences with even partial homology can be used as hybridization probes.

• mRNA transcript size can be detected.

• RNA splicing is visible because alternatively spliced transcripts can be detected.

• The cost of running many gels is low once the equipment is set up.

• Blots can be stored for several years and reprobed if necessary.

• Quantity and quality of RNA can be easily verified after electrophoresis and before further processing is done.

THANK YOU