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An overview of northern blotting.. with its applications, and pros and cons
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NORTHERN BLOTTING
IQRA JABBAR
M.PHIL.
SCHOOL OF BIOLOGICAL SCIENCES
WHAT IS BLOTTING?
Proteins, DNA or RNA, can be transferred onto a carrier
nitrocellulose or nylon membrane
The transfer of biological samples from a gel to a membrane and their subsequent detection on
the surface of the membrane
Blotting Types
Southern(DNA)
Northern(RNA)
Western (Protein)
TYPES OF BLOTTING
NORTHERN BLOTTING
A technique to detect and measure specific
RNA sequences
Developed by James Alwine and George Stark
at Stanford University in 1977
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Oligo (dT) column chromatography can be done to isolate only polyA+ RNAs
Formaldehyde used as denaturing agent in gels to remove secondary structures.
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Nylon membranes are preferred • positively charged• high capacity to bind
nucleic acids• greater robustness on
handling)
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cDNA, RNA, or oligonucleotides with a minimum of 25 complementary bases to the target sequence.
Probes can be Radioactive (Radiolabelled with P32) Or Chemiluminiscent (probe attached to enzyme for chemiluminiscent substrate or to a ligand..)
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Steps involved in northern blotting
BLOTTING SET UP
Transfer buffer contains formamdie• It lowers the annealing temperature for probe-
RNA • Prevents RNA degradation at high
temperatures.
CHEMILUMINISCENT PROBES
APPLICATIONS OF NORTHERN BLOTTING
To observe a particular gene’s expression
pattern between
Tissues Organs Developmen
tal stages
Environmental stresses
Pathogen infection
APPLICATIONS OF NORTHERN BLOTTING
Comparison of
expression of
oncogenes and tumor
suppressor
genes between
cancerous and normal
tissues
Normal Cancerous
Oncogene
Tumor suppressor
gene
APPLICATIONS OF NORTHERN BLOTTING
By using only one probe.. If obtain
variance in the level of each
band….
• May be alternative spliced
products
• Or repetitive sequence motifs
• Deletion or errors in transcript….
Can be tested by changing
probe target sequence… find the
missing regionSingle probe is used for detection
ADVANTAGES
High specificity
Quality and quantity of RNA can be determined
Probes with partial
homology can be used
Membranes can be
stored and re-probed
DIS-ADVANTAGES
Dis-advantages
Small number of genes can be
dealt at one time
Degradation by RNases
Use of harmful chemicalsLow sensitivity
Detection with multiple probes is
difficult
Thank you