Class Notes (811,481)
United States (314,602)
Boston College (3,526)
Biology (470)
BIOL 5060 (27)
Hoffman (27)
Lecture 8

BIOL 5060 Lecture 8: 2:6_RecombinantDNATech_Hoffman

7 Pages
Unlock Document

BIOL 5060

2/7_RecombinantDNATechnology_Hoffman 2/7/2017 2:11:00 PM Value of a Genomic Library • Library is a collection of clones • Different insert sizes per clone • Each represents a collection of clones that in total, represent the sequence of the genome cDNA libraries • Most come from fish and yeast but can insert human, frog, or mammalian cDNA and insert it into a fish and yeast expression vector Why cDNA? • ** Allows for expression of a gene from a foreign organism; allows for determination of the ORF • Final product will have… o 5’ un-translated region (UTR)—ORF—3’ UTR—AAAAA ▪ AAAAA= (poly A sequence that was in the RNA) o Allows for cloning in a vector that supplies a promoter that allows this gene to be expressed • The cDNA itself doesn’t contain a promoter region- need a vector • Need to make sure we clone it in the right direction (in the way the promoter is driving transcription) o Don’t want it backwards • How can we do this? Trick to directional cloning of cDNA… o 1) Start w mRNA poly dt oligote (TTTTTT) allows for poly- synthesis ▪ Along w poly dt, has and XhoI site ▪ TTTTT matches poly A tail (AAAAA) on mRNA o As long as the 3’ end base pairs we can polymerize; the 5’ end can be flapping in the wind (we don’t care) ▪ 5’ has XhoI—services second strand synthesis o 2) Reverse transcribe with dNTPs (methyl-dCTP) ▪ All C nucleotides methylated o 3) Second strand synthesis- also has methyl-dCTP ▪ All C nucleotides on second strand will be methylated ▪ Now a double stranded DNA o 4) Ligate on EcoRI linkers (linker= short double stranded DNA with restriction enzyme site) ▪ Get added to 5’ end o 5) Blunt end ligation- cut with EcoRI and XhoI ▪ these don’t cut fully methylated sites ▪ un-methylated linkers are cut leaving Sticky 5’ end ▪ XhoI is hemi (half)- methylated… why? We ordered it… it doesn’t have methyl groups on the C—wasn’t created by us; we bought it from scientist  Hemi-methylated DNA is still cut, fully methylated is not ▪ SO we do cute the XhoI site XhoI sticky end on the other side, too o 6) Insert gets ligated in a directional fashion ▪ with XhoI side distal from the promoter Plasmid Libraries= much more common Re-amplifying a plasmid library • Goal is to get a prep of DNA that has the same collection of clones as the original library (you want to avoid losing clones) • 1) need to know how many clones are present • 2) Generate 10x as many transformants o depending on probability—distribution ▪ outcome is yes/no o if we have 4x as much we lose 2% of clones, if we have 5x as much we lose .7% as much ▪ if we do 10x as much, almost every clone will be protected • 3) Plate for a subconfluent number of colonies per plate o Not a solid lawn of transformants o Some plasmids will reduce growth plate- we don’t want to select against them because they might represent genes we are interested in o You want a plate where colonies are easily distinguishable and you can see lighter areas of growth= space for slower growing colonies so they can catch up Screening Libraries Based on… • 1) Expression of a biologically active product- Functional Cloning • 2) Detection of a physical molecule (either a specific seq. of DNA [Hybridization] or a protein [immune detection, other metals- in vitro activity]) Functional Cloning (expression of a biologically active product) • 1) Complementation of a recessive mutation in a host organism o Mutant host, wild-type clone gene, detection= phenotypic shift from mutant to wild-type phenotype • 2) Multi-copy effect of a gene o Mutant host, wild-type clone gene (but not defective gene), again looking for (detection) mutant to wild type phenotype o Wild type host, wild type clone gene, detection: wild type to mutant phenotype
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.