chapter 10 Flashcards
recombinant DNA technology (RDT)
methods for obtaining, amplifying, and manipulating DNA fragments
how are DNA molecules cut
by restriction enzymes at the cut site to generate sticky or blunt ends that are later used for DNA insertion into the plasmid
restriction enzymes (REs)
cut DNA strands by breaking covalent (phosphodiester) bonds of backbone
recognition sites
specific DNA sequences that REs bind to (usually downstream to promoter region)
cut sites
where REs cut DNA
sticky (overhang) ends
single strand with complementary base pairs, 5’ overhand
blunt ends
have no overhand
EcoR1
restriction enzyme in e.coli that generates sticky ends
palindromic sequence
cut sites are usually palindromic, most recognized by RE
why did REs evolve (3 reasons)
- occur naturally in many prokaryotes
- defense against phages
- modify their own DNA to prevent self digestion
joining the DNA molecules
DNA ligase joins the inserted DNA into the vector/plasmid
DNA ligase
joins two DNA molecules by covalent linkage of deoxyribose backbone (can join blunt with sticky ends if there’s a 5’ phosphate and 3’ OH in the gap)
insertion of donor DNA into the plasmid (3)
1) Donor and plasmid DNA are cut with compatible REs (i.e their ends are compatible)
2) sticky ends hybridize because of base pairing but there’s a gap in backbone (no phosphodiester bond)
3) ligase seals gap in backbone
replication and amplification of DNA
recombinant plasmid DNA is introduced into host cells through transformation where it’s replicated
amplification’s 2 mechanisms
1) all cells in culture have 1 copy of plasmid
2) many plasmids maintained in multiple copies per cell
only circular DNA molecules replicated ____, linear DNA must ____
1) autonomously
2) integrate into host chromosome
isolating specific sequence approaches (2)
library method and PCR amplification of specific sequence
library method
DNA fragments carried on plasmids or in phages are amplified by e.coli and screened for desired sequence
PCR amp of specific sequence
DNA polymerase used with specific primers to synthesize specific fragments
cloning
isolating and amplifying particular sequence (finding sequences corresponding to specific gene and amplify by plasmid insertion)
cloning with library method
break genome into small gene sized pieces (creating LIBRARY in cloning vector) and identify piece of interest with detection method
cloning vector
carries foriegn pieces of DNA to be replicated and amplified in cell systems
4 basic cloning vectors
plasmids, lambda phage, fosmids, and artificial chromosomes
plasmid vectors
most convenient, small, easy manipulation
bacteriophage vectors
allows larger inserts, easy to infect into bacteria and high level protein production
fosmid and artificial chromosomes (YACs and BACs)
can carry largest inserts
all plasmids have (4)
- origin of replication (determines copy number)
- polylinker/MCS (multiple cloning vectors with unique cut sites)
- selectable marker (in case transformation is insufficient)
- means to detect inserts (in case ligase insufficient)
types of libraries (2)
genomic fragments and cDNA
genomic fragments
made by randomly breaking genome, represents entire genome
cDNA
each vector has single gene or gene frag, ONLY protein coding sequences (unequal rep)
making genomic library (3)
- digest genomic DNA and plasmid vector with same RE
- mix digested plasmid and genomic DNA (joined with ligase)
- transform into e.coli
making cDNA (5)
1) lyse cells and extract total RNA
2) putify mRNA using olig(df) column
3) use reverse transcriptase + olig(df) primer to make SS cDNA
4) use oligonucleotide primer and DNA polymerase II to synthesize complementary strand
5) ligate resulting duplex cDNAs into cloning vector
cloning methods
probes, complementation
probes
molecules designed to recognize library vectors with specific sequence on basis of structure
