lecture 17 Flashcards
recombinant DNA technology
ability to manipulate DNA
REs (restriction endonucleases)
“DNA scissors” that cleave DNA at specific sequences
most recognition sequences are 4-6 bp long and palindromic
cuts can be blunt or leave a sticky overhang
gel electrophoresis is used to
separate DNA of different sizes
hybridization is the process of
DNA renaturation
how does hybridization work?
increasing temp can denature the DNA to release single strands
lowering temp can cause strands to renature
southern blotting
the transfer of DNA to a membrane and hybridization w a labeled probe
northern blotting
same process as southern blotting but w RNA
DNA cloning
producing many identical copies of a DNA sequence
recombinant DNA
DNA molecule w DNA from many sources
plasmid
small circular DNA molecule used in bacteria
plasmids can be introduced into bacteria through
transformation
when the bacteria has taken up the plasmid, when they replicate..
the plasmid is also replicated so you end up w a lot of copies
genomic library
representative of all the genomic sequence of an organism
-includes coding and noncoding DNA
-library will be the same regardless of cell type
cDNA library only contains
genes transcribed into mRNA
4 steps to make recombinant DNA
- cut the DNA of interest
- cut the plasmid vector
- insert DNA into the plasmid
- reseal nicks w DNA ligase
3 steps to create a genomic library
- the genome is digested using REs or DNA shearing
- the DNA fragments are cloned into plasmids
- plasmids are introduced into E coli as hosts
5 steps to create a cDNA library
- total RNA is extracted from organism, isolate mRNA
- cDNA (complementary) is prepped using reverse transcriptase using a polyT primer complementary to the polyA tail of mRNA
- the cDNA is inserted into a vector (plasmid) and cloned to produce a cDNA library
- cDNA can be inserted into a vector
- introduce vector to bacteria
PCR
polymerase chain reactor
= a very powerful technique, allows you to make billions of copies of a nucleic acid
v sensitive, fast and easy
3 steps for PCR
- heat to separate strands
- cool and anneal primers that flank the sequence of interest
- allow DNA polymerase to extend from the primer
***this is repeated 30-35 times!
why do we need a thermostable DNA polymerase for PCR?
if it wasn’t, the DNA polymerase would denature when heated!
we would need to add a fresh one after each cycle!
PCR uses
- used while generating DNA libraries to amplify segments being cloned
- used to detect small amounts of DNA/RNA from a pathogen
- can be used to amplify STRs to be used in DNA fingerprinting
sanger sequencing makes use of
ddNTPs (dideoxyribonucleoside triphosphates)
why is DNA polymerization impossible is a ddNTP is incorporated?
ddNTPs have a 3’ H instead of a 3’ OH which makes DNA polymerization impossible
DNA microarray
a slide has hundreds of DNA fragments in diff spots that are complementary to mRNA for diff genes
RNA seq
RNA from a cell is isolated and then sequenced
what is the advantage of RNA seq over microarray?
don’t need to know sequence of mRNA ahead of time for RNA seq
in situ hybridization uses a
labeled single strand DNA or RNA probe that targets a specific sequence in a cell
a gene encoding a protein of interest is fused to a
reporter gene
reporter gene=
a gene that encodes a protein product that can be easily modified
gene knockout
completely eliminating a gene leads to a gene knockout which can be used to simulate disease conditions
