Chapter 12 Flashcards
Genetic engineering
the use of in vitro techniques to alter genetic material in the lab
First gene clone and purified from pigs
insulin
Recombinant DNA technology
the artificial recombination of DNA from two organisms
Restriction Endonuclease Enzyme
- recognition specific DNA sequences and cut DNA
- essential for in vitro DNA manipulation and gene cloning
Methylation
cells must protect themselves from inadvertent destruction
Type II
- cleave DNA within recognition sequence
- most useful for specific DNA manipulation
Type I
- first discovered/purified
- cuts DNA at random far from recognition sequence
- little practical value
Type III
- large
- cleaves outside of recognition
- rarely used
How do structures of cleaved products differ?
3’ or 5’ overhang or blunt ends
recognition sequence are usually….
4-8 bp long
how are the 3 letters in REs designated?
- first letter is genus RE isolated
- next two represent species of RE isolated from
- Roman numeral indicated order of discovery
Escherichia coli enzyme designation
EcoRI, EcRV
Escherichia coli recognition sequence
I: G|AATTC
V: CTTAA|G
DNA fragments produced by EcoRI
5’ overhangs
DNA fragments produced by Pstl
3’ overhangs
DNA fragments produced by Smal
blunt ends
The nucleic acid sequence where EcoRI cuts
GAATTC
Molecules with complimentary sticky ends can easily….
anneal or form H bonds
Ligation
by DNA ligase can rejoin two sugar phosphate backbones of DNA through covalent bonding
After modification, DNA can no longer….
be cut by a RE
3 main steps of gene cloning
- isolation and fragmentation of source DNA
- insertion of DNA fragments into cloning vector
- intro of cloned DNA into host organism
plasmids
natural vectors and have useful properties of cloning vectors
why are plasmids useful?
- small
- independent origin of replication
- multiple copy # so multiple copies of cloned genes
- presence of selectable markers
vector transfer to host can be accomplished by
chemical transformation of host cell, sometimes conjugation or transduction, or electroporation
essential features of plasmid pUC19
- ampicillin resistance, polylinker with multiple restriction enzyme cut sites,
- lacZ is fully functional even with presence of polylinker
pUC19 size
- small
- 2686 bp
enzyme used for cloning
- restriction endonuclease
- DNA ligase
- reverse transcriptase
- DNA polymerase
DNA ligase
catalyzes the going of two strands of DNA between 5’ and 3’ adjacent nucleotides with either cohesive or blunt ends
reverse transcription
converts RNA into DNA
DNA polymerase
mostly used for 5’3’ polymerizing activity
- also have 3’5’ and 5’3’ exonuclease activity
blue colonies
do NOT have vector with foreign DNA inserted
white colonies
have foreign DNA inserted
genomic library
nearly/complete copy of organisms genome contained as recombinant DNA plasmids/ phages
To clone only expressed genes, libraries can be constructed using….
organisms mRNA
how is mRNA cloned?
not directly.. used as template for retroviral reverse transcriptase
common host strains
E. coli, Bacillus subtilis, saccharmoyces cervisiae
ideal hosts should be….
- rapid growth
- non pathogenic
- incorporates DNA
- stable
- allows replication of vector
E. coli
pro: well developed, main strains, well know
cons: pathogenic, traps proteins
bacillus subtilis
pros: easily transformed, nonpathogenic, secreted proteins
cons: unstable, less developed
saccharomyces cerevisiae
pros: well developed, nonpathogenic, processes mRNA, easy to grow
cons: unstable, doesn’t replicate bacterial plasmids
Agarose Gel Electrophoresis
separates DNA by charge/size
Agarose gels can be stain with ______ and DNA is visualized under _______.
ethidium bromide
UV light
the same DNA cut with different restriction enzymes will have _____ banding patterns on an agarose gel.
different
RE map
a map of location of restriction enzyme cuts on a segment of DNA
PCR
Polymerase Chain Reaction
- DNA replication in a test tube
- rapid amplification in number of copies of specific DNA
applications of PCR
- determine DNA from a particular region
- cloning specific fragments
- identifying DNA source (crime)
- paternity tests
- ancient DNA compared to modern
- ascertaining difficult to culture organisms
steps in PCR amplification
- denatured by heating
- synthetic DNA is added
- DNA polymerase is added (Taq poly/ Pfu poly)
- heat & cool
a variation of PCR
reverse transcriptase PCR
- add primer and reverse transcriptase
- form single stranded cDNA
- add RNase H
- add primer specific to 5’ end + taq = double stranded cDNA
nucleic acid hybridization
base pairing of single stranded DNA/RNA from 2 different sources to give hybrid double helix
nucleic acid probe
segments of single-stranded DNA that is used for hybridization
*ss DNA complementary to the sequence of gene of interest
how are nucleic acid probes created?
cloning, synthesis, denaturing, or fragment of DNA
molecular beacon
radioactive, fluorescent dye
synthesized DNA is used for…
primers and probes, and in site-directed mutagenesis
FISH
Fluorescent In Situ Hybridization
-fluorescent probes attach to Olig.
Southern blot
- labelled DNA probes is used to find complementary sequence by hybrid. with radioactive probe
- position of hybrid band is noted using X-ray/radiography or fluorescent
- only some DNA fragments hybridize.
Northern blot
- RNA is in the gel
- radioactive probe to specific gene
- same blot probed with 5S RNA as a control
how to detect clones containing correct DNA inserts
- must establish that cloning procedures worked
- initial selection– antibiotic resistance; white-blue screening
- if cells express foreign gene, expression might be detected
- look for specific DNA id the gene is not expressed.
reporter genes
encodes proteins easy to visualize
i.e. lacZ, luciferase, green fluorescents
gene fusions
promotors or coding sequences of gene of interest can be swapped with those of reporter genes to elucidate gene regulation under various conditions
site-directed mutagenesis
performed in vitro and introduces mutations at a precise location
cassete mutagenesis/knockout mutations
DNA fragment can be cut, excised and replaced by synthetic DNA fragments
gene disruption/insertional inactivation
cassettes (i.e. antibiotic resistance genes) are inserted into a gene and disrupting its function
knockout mutation
total loss of gene function when disrupted gene recombines into chromosome
