HMB265H1 Lecture Notes - Northern Blot

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Published on 9 Feb 2012
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-DNA from the organism extracted & cut into fragments with enzymes. Fragments
separated into groups of fragments of different sizes (fractionated) by using
electrophoresis. Fragments are blotted onto a piece of porous membrane where they
maintain the same relative positions = SOUTHERN blot -> probe for SPECIFIC DNA
-Southern Blot: the transfer of electrophoretically separated fragments of DNA from a
gel to an absorbents sheet such as paper; this sheet is the immersed in a sol’n
containing a labeled probe that will bind to a fragment of interest
-heat DNA -> separate strands, the membrane is placed in a sol’n of the probe. The
single-stranded probe will find & bind to its complementary DNA sequence. On the blot,
this binding concentrates the label in one spot -> relevant DNA on gel is revealed
-Find SETS of GENES -> use DNA microarray -> “genome wide probing”; array of DNA
fragments representing all the genes in a genome can be glued to the surface of a glass
slide = MICROARRAY. Probes used are usually complex mixtures made by converting
mRNAs from one tissue into DNA (cDNA). Bathed microarray in labeled probe where
they bind -> which genes are being transcribed in the tissue
-use PCR (polymerase chain reaction) to detect homologs of a region of an unknown
sample that has been sequenced: pick 2 short single stranded DNA segments that flank
the region in question (used as primers to initiate DNA replication) -> get newly
synthesized copies of the unknown sample that in turn act as templates -> get multiple
copies of DNA segment that is equivalent of that region .
primers only work if unknown sample contains a homolog of the target region in
question.
If PCR obtained = DNA present in the sample.
Rapidly detect the presence of specific segments of interest in any type of diagnostics
(need large amt to be detected)
-Detect an RNA transcript in a specific tissue -> extract all the mRNA & separate into
garments of different sizes (use electrophoresis) -> blot onto membrane -> use cloned
gene as a labelled probe to highlight the mRNA if it is present = NORTHERN BLOT
-Northern Blot: the transfer of electrophoretically separated RNA molecules from a gel
onto an absorbent sheet which is then immersed in a labeled probe that will bind to the
RNA of interest
-Probe for specific protein -> use antibodies as probes (made from an animal’s immune
system -> have high affinity to specific protein which acts as an antigen). Protein
mixture extracted from cells -> separate into bands via electrophoresis -> blot onto
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Document Summary

Dna from the organism extracted & cut into fragments with enzymes. Fragments separated into groups of fragments of different sizes (fractionated) by using electrophoresis. Fragments are blotted onto a piece of porous membrane where they maintain the same relative positions = southern blot -> probe for specific dna. Heat dna -> separate strands, the membrane is placed in a sol"n of the probe. The single-stranded probe will find & bind to its complementary dna sequence. On the blot, this binding concentrates the label in one spot -> relevant dna on gel is revealed. Find sets of genes -> use dna microarray -> genome wide probing ; array of dna fragments representing all the genes in a genome can be glued to the surface of a glass slide = microarray. Probes used are usually complex mixtures made by converting mrnas from one tissue into dna (cdna).

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