CAS BI 216 Lecture Notes - Lecture 6: Hybridization Probe, Complementary Dna, Reverse Transcriptase

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I. Libraries - isolate sequences of genome and put them into individual vectors to find
specific
A. Genomic
1. All
2.
B. cDNA
1. Requires gene that is Transcribed
2. Each gene transcribed at own rate
3. Rna is proportionate to amount of expression
C. mRNA
1. Has polyadenalated tail
2. Can use tag to pull out mRNA using a column with poly dT that will base
pair to A or directly mixing with
a) Will get mRNA, and then synthesize complementary DNA using
reverse transcriptase found in retrovirus
b) Makes complementary DNA - creates cDNA and mRNA double
strand, digest mRNA, and then create complementary
c) Put cDNA in vector to create library
D. Genomic vs cDNA library
1. Genomic has intron, exon, gene, etc
2. cDNA - only exons and based on amount of RNA, wont have promotors
a) If Gene A<B and B<C, cant state relationship btw A and C yet
II. How do we find the clone of interest from a library?
A. Know human cystic fibrosis gene, but want mouse one
B. Depend on hybridization - similar human and mouse gene
1. Labeled DNA probe - Take library and replicate using that sticking onto
new paper method - lyse it with bases or sponge and DNA will stick to
filter exactly where colonies were - incubate with radioactive label to CF
gene and radioactive signal only from CF gene
2. Separate strands, add radioactive DNA, binds to target
C. How do you do it if you dont know the sequence at all (alteRNAtives)
1. DNA sequence derived from a purified protein
a) Know some of the amino acids in the protein - reverse translation
based on which codons code for those amino acids (many
possibilities) - made a degenerate probe that contains these
possibilities
2. DNA sequence derived from a sequence related to the one of interest
a) Use evolutionary conserved protein - proteins from diff species
that do similar things and have similar AA sequences
b) Generate protein probe without purifying the protein using the
conserved sequences
III. Computational analysis of DNA sequence
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