Lecture 13: Biotechnology Flashcards
genetic engineering
in vitro techniques to alter genetic material from prokaryotes
where does biobrick assembly occur?
test tube
heterologous expression
expressing a gene in a different host
PCR
rapid amplification in DNA sequence copies
thermocycler
automated PCR machine
Quantitative PCR
amplifies RNA after conversion to DNA
PCR steps
- denaturation
- flanking/binding
- add DNA polymerase
- Heat & cool
reverse transcription PCR
makes DNA from mRNA template
what does reverse transcription PCR do?
produces intron free eukaryotic gene
what end is the primer added to in PCR?
5’
Gel electrophoresis
separates DNA based on size & charge
what electrode do nucleic acids migrate to?
positive
what size of molecules move faster in gel electrophoresis
small molecules
what does ethidium bromide do in gel electrophoresis?
is used to visualize the DNA fragment bands
will the same DNA cut with different restriction enzymes have the same or different banding patterns?
different
nucleic acid hybridization
DNA / RNA from 2 diff sources form a hybrid double helix
nucleic acid probe
segment of DNA strand that has a predetermined identity
southern blot
DNA is in the gel & probe is RNA/DNA
northern blot
RNA is in the gel & the probe is DNA/RNA
synthetic DNA usage
used for primers, probes & site directed mutagenesis
what is synthetic DNA made of
oligonucleotides of 100 bases
molecular cloning
movement of a gene from original source to small/manipulable genetic element
what does molecular cloning result in?
recombinant DNA
recombinant DNA
molecule containing DNA from different sources
what 4 enzymes are used for cloning?
- restriction endonucleases
- DNA ligase
- reverse transcriptase
- DNA polymerase
what does DNA ligase join together
joins 5’ phosphate & 3’ OH of adjacent nucleotides
DNA polymerase
5’ to 3’ activity
what do restriction endonucleases do
cuts DNA
where are restriction endonucleases more commonly found
prokaryotes
Type II restriction endonucleases
cleaves DNA within sequence
- most useful
- palindrome
Type I
cuts DNA randomly & not within sequence
Type III
cleaves outside of sequences
recombineering
allows foreign DNA to be inserted into vectors/chromosome
what was recombineering originally developed in?
saccharomyces cerevisiae
where do cloning vectors replicate
the host
why are plasmids used as cloning vectors?
small size & they have selectable markers
pUC19 uses
insertional inactivation
insertional inactivation
lacZ is inactivated & B-galactosidase is not produced
blue vs white colonies
blue: vector is not inserted with foreign DNA
white: vector is inserted with foreign DNA
PCR cloning vectors
plasmids specifically for cloning DNA products made by taq
where are shuttle vectors replicated & maintained
replicate & be maintained in E. coli & yeast
yeast artificial chromosomes (YACs)
chromosomes that clone directly into yeast
properties of a good host for cloning
- non-pathogenic
- rapid growth
electroporation
electric pulse is applied to form pores
microinjection
gene gun inserts into cells
expression vectors
vectors that control expression of genes
why are eukaryotic genes that contain introns easy to isolate?
because of poly (A) tails
fusion proteins
join target & carrier proteins
site directed mutagenesis
introduces mutations at a precise location
what can site directed mutagenesis be used for?
assess amino acids activity in a protein
where does site directed mutagenesis occur?
in vitro
how many base DNA oligonucleotides can be obtained through site directed mutagenesis
12 - 40
cassette mutagenesis
synthetic fragments that can change base pair changes
gene disruption
inserts cassettes in middle of a gene
knockout mutation
loss of function of a gene in which cassette is inserted
reporter gene
encodes protein easy to detect & assay
example of a reporter gene
green fluorescent proteins
operon fusions
signals are fused with transcriptional signals of another gene
protein fusions
2 proteins are fused & share same transcriptional & translational start/stop
what do protein fusions yield?
1 hybrid polypeptide
genome editing
uses CRIPSR from streptococcus pyogenes to alter eukaryotic genomes