bioinformatics Flashcards
BLAST
basic local allignment search tool
blastn/nucleotide blast
search all DNA sequences in database for
a significant match to the DNA sequence you are analyzing
blastx
search all protein sequences in database for
a significant match does it code for protein
for any given sequence there are _ possible reading frames
6, 3 on each strand of dna
when you are using blast you input for dna in to the
query
blastp/protein blast
input protein sequence and search protein
databases for significant match to any other known
protein sequences
what can blastp help determine
if there is an ortholog for a gene
what does % identities and % positives mean in blastp
% identities = % of amino acids that are in the same position
% positives includes exact matches and amino acids of similar chemical properties
expression vector
cloning vector that contains a regulated bacterial promoter next to the cloning site
what are the step to making recombinant proteinn
1)Isolate mRNA from 2)Treat with reverse transcriptase to
synthesize cDNA
3)Ligate cDNA’s into pUR expression
vector & transform E. coli to create
cDNA library
4)Sequence random clones until you findone that has an Open Reading Frame that matches the known protein sequence
what is recomb. technology good for
making insulin, human groth factor safe virus for vaccines
when making transgentic animals where do you inject the recombinant DNA
in the mothers oocyte (in vitro fertilization)
what is the first step of creating a transgenic animal
clone cDNA into a expression vector
Transgene =
a segment of DNA containing a gene sequence that has been isolated from one organism and introduced into a different organism.
what are the main questions asked when looking at Gene Expression
(1) When during development is gene transcribed?
(2) In what tissues is the gene transcribed?
(3) In what cells is the gene transcribed?
when using a northern blot you can determine
when during development is gene transcribed?
what is the first step when doing a northern blot
Extract mRNA from each sample & run out on an agarose gel
what is the order/components in a petri dish when making a northern blot filer
there is a buffer that the gel (from electroleysis ) sit in on top of the gel there is a nylon membrane, on top of the membrane are some paper towels for weight
how does the RNA get transferred onto the membrane filter
As buffer is drawn up by capillary action, the paper towels soak up the buffer and the RNA in the gel is transferred
after the RNA is on the filter paper what helps you find the gene of interest
a probe - made by recomb. dna w/t radioactive neucloides
Now incubate membrane with _____ probe. Probe DNA
strands will to _______ complementary RNA on gel
denatured,hybridize
Overlay northern blot filter with -________. Radioactivity will expose film and ______ will be seen
X-ray film,dark bands
the techique that can best help you find out what tissues a gene is being expressed is
situ hybridization
situ hybridization:
Make a labeled “probe” from the recombinant DNA plasmid and hybridize to embryos that have been fixed and permeablized.
FISH = Fluorescence In Situ Hybridization is
technique is
to localize cloned DNA sequences to a specific
chromosomal site.
Mapping the gene sequence
how does FISH work
fluorescent probes that
bind to only those parts of the chromosome with which they show a high degree of sequence
similarity
