Class Notes (807,339)
United States (312,494)
Boston College (3,489)
Biology (467)
BIOL 5060 (27)
Hoffman (27)
Lecture 10

BIOL 5060 Lecture 10: 2:16_recombinantDNATech_Hoffman

5 Pages
Unlock Document

Boston College
BIOL 5060

2/16_RecombinantDNATechnology_Hoffman 2/16/2017 3:02:00 PM Real Life example- Gene 1 inside Gene 2 st • 1 thing we do= make sure we have a wild type gene • PCR amplify= 1 way to get to a purified reagent without having to go through a library (if we know the planking regions) • 80-mers • You could kind of see stuff but why did it come out pretty crappy? o Even though each even has a 99% accuracy, after doing it 80 times at a 99% accuracy rate, your product will not be very similar to your starting product o The 80-mer unpurified reagent is heterogeneous in terms of the 5’ end doesn’t effect PCR reaction but DOES effect the sequencing reaction o Error in the chemical efficiency of adding a nucleotide… only 99.3% of them get a nucleotide ▪ Failing to put a nucleotide changes the length of the whole thing ▪ Could have used a better oligote for this sequencing BLAST Query (what u start w.) Subject (what u find) • BLASTN Your DNA seq. DNA seq. in DNA database o Used for your sequence in the database…. Did you clone something that has already been sequenced? Is it related to something in the database? o Because of the complexity of DNA you need a good degree of alignment for the computer to think they’re related ▪ Better at finding things that are identical • BLASTX Your DNA seq. A protein in the protein database o Translate tool—your product depends on the DNA’s capacity to encode o Protein can be in the + reading frame (going left to right) or it can be going backwards (right to left) in the – reading frame ▪ ….6 possible reading frames +1, +2, +3 OR -1, -2, -3 ▪ Reading frames defined by triplets (codons) o What is the potential coding ability of your DNA sequence and how does that relate to the protein database? o Proteins are even more complex- there are 20 possible amino acids (residues) at each site (1/20 chance of two strands having the same AA vs. ¼ chance of 2 strands having same NT in DNA) ▪ Proteins can align much more distantly o Because proteins are more complex, its better at finding related sequences o Only works for coding region- it will jump over introns • BLASTP Your protein A protein in the database o Need to pay attention to the quality of the alignment • You get an E value= tells how likely this is to be significant o Based on the 1) degree of identity and similarity o 2) Length of the alignment o 3) Lower the E value the better the alignment- means its more improbable that it was just a random alignment o 4) 10 ^ -50 or less is thought as suggesting biochemical conservation—suggests protein is of a protein class ▪ shorter protein needs better conservation When you clone something and want to know what you have, you sequence (BLAST) can lead to hypothesis regarding the function of what you have… In Vitro Mutagenesis- a way of generating mutant alleles (altered sequences of DNA) to ask about the biological relevance • Can be used in… • 1) Forward Genetics- what mutat
More Less

Related notes for BIOL 5060

Log In


Don't have an account?

Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.