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 organism
Restriction Endonuclease Enzymes
- recognize specific DNA sequences and cut DNA
- essential for in vitro DNA manipulation and gene cloning
methylation
Cells must protect their own DNA 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 sequences
- rarely used
How do structures of cleaved products differ?
3’ or 5’ overhang or blunt ends
Recognition sequences are usually….
4-8bp long
How are the 3 letters in REs designated?
- first letter is genus RE isolated from
- next two represent species RE isolated from
- roman numeral indicated over of discovery in that species
Escherichia coli enzyme designation
EcoRI, EcoRV
Escherichia coli recognition sequence
I: G|AATTC
V: GAT|ATC
DNA fragments produced by EcoRI
5’ overhangs
DNA fragments produced by PstI
3’ overhangs
DNA fragments produced by SmaI
blunt ends
The nucleic acid sequence where EcoRI cuts
GAATTC
Molecules with complementary sticky ends can easily…
anneal or form H bonds
Ligation
by DNA ligase can rejoin two sugar phosphate backbones of DNA through covalent binding
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 fragment into cloning vector
- introduction of cloned DNA into host organism
plasmids
natural vectors and have useful properties are cloning vectors
Why are plasmids useful
- small
- independent origin of replication
- multiple copy # so multiple copies of cloned gene per cell
- 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
Enzymes used for cloning
- restriction endonuclease
- DNA ligase
- reverse transcriptase
- DNA polymerase
DNA ligase
catalyze joining of two strands between 5’ phosphate and 3’ OH of adjacent nucleotides
Reverse transcriptase
converts RNA into DNA
DNA polymerase
Mostly used for 5’3’ polymerizing activity; may 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
a nearly complete/complete copy of an organisms genome contained as recombinant DNA plasmids or phages
To clone only expressed genes, libraries can be constructed using…
the organisms’ mRNA
How is mRNA cloned
It cannot be cloned directly and is used as a template for retroviral reverse transcriptase
common host strains
E coli, bacillus subtitles, saccharomyces cerevisiae
Ideal hosts should be…
- rapid growth in inexpensive medium
- nonpathogenic
- capable of incorporating DNA
- genetically stable in culture
- equipped with appropriate enzymes to allow replication of the vector
E coli
- well developed genetics
- many strains
- best known bacteria
- potentially pathogenic and periplasm traps proteins
bacillus subtilis
- easily transformed
- nonpathogenic
- secretes proteins
- endospore formation simplifies culture
- genetically unstable and genetics less developed
saccharomyces cerevisiae
- well-developed genetics
- nonpathogenic
- can process mRNA and proteins
- easy to grow
- plasmids unstable and will not replicate most bacterial plasmids
Agarose Gel Electrophoresis
- separates DNA molecules based on size
- uses electrical field to separate charged molecules; nucleic acids migrate towards positive electrode
Agarose gels can be stained with ___________ and DNA is visualized under _________.
ethidium bromide, UV light
The same DNA cute with different restriction enzymes will have ___________ banding patterns on an agarose gel.
different
RE map
a map of the location of restriction enzyme cuts on a segment of DNA
PCR
DNA replication in a test tube - DNA amplification
-rapid amplification in the # of copies of specific DNA sequences for further analysis
Applications of PCR
- determing DNA sequences
- cloning a specific fragment
- identifying DNA source from a crime scene
- paternity determination
- analysis of ancient DNA
- organisms presence in a specimen
Steps in PCR amplification
- DNA denatured by heat
- add DNA pol
- Ad Taq pol
- heat and cool many many many times in similar cycles
A variation of PCR
reverse transcriptase PCR
Nucleic Acid Hybridization
Base pairing of single strands of DNA or RNA from 2 different sources to give a hybrid double helix
nucleic acid probe
segment of single-stranded DNA used in hybridization and has a predetermined identity
How are nucleic acid probes created?
By cloning, synthesis, or denaturing a fragment of DNA
molecular beacon
label probes
Synthesized DNA is used for..
primers and probes, and in site directed mutagenesis
FISH
Fluorescent In Situ Hybridization; uses fluorescent probe attached to oligonucleotide
Southern Blot
a hybridization procedure where DNA is in the gel and probe is RNA or DNA
Northern Blot
RNA is in the gel; Radioactive probe to a specific gene to total RNA which is run on a gel
How to detect clones containing correct DNA inserts
- establish if cloning procedure worked
- antibiotic resistance/blue-white screening
- if cells express the foreign gene
- look for specific DNA is the gene is not expressed
reporter genes
- encode proteins that are easy to visually detect and assay
- lacZ, luciferase, green fluorescent protein
gene fusions
promoters or coding sequences of genes 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
-can be used to assess activity of specific amino acids in a protein
cassete mutagenesis/knockout mutations
DNA fragments can be cut, excised, and replaced by a synthetic fragment
gene disruption/ insertional inactivation
When cassettes are inserted into a gene disrupting its function
-may cause knockout mutations
knockout mutations
total loss of gene function; great for studying what genes do
