Recombinant DNA and Genomics Flashcards
recombinant DNA is DNA that is created by
joining together pieces of DNA from various sources
recombinant DNA technology is also called
gene splicing
clones are
recovered copies of recombinant DNA molecule
clones are used to
study structure and orientation of DNA
two important tools to construct and amplify DNA molecules are
restriction enzymes and cloning vectors
steps to recombinant DNA
- Cut DNA of interest and vector with restriction enzyme.
- Incubate together (or cut together).
- Anneal cut DNA (hydrogen bonds).
- Ligate nicks in DNA backbone (covalent bonds).
- Transform vector into host.
- Screen for host with appropriate vector.
- Do rad science
restriction enzymes cut DNA in what types of ends
stick (cohesive) and blunt ends
what is a restrictive enzyme that has been identified in e-coli?
ecoR1
DNA ligase
joins fragments sealing the phosphodiester backbone
cloning vectors are
DNA molecules that accept DNA fragments
distinguishable characteristics of cloning vectors
restriction enzyme sites
selectable gene markers
restriction enzyme sites allow
insertion of DNA fragments
the selectable gene marker is used to distinguish
host cells that have taken up the DNA fragments from those that have not
a successful cloning vector includes
- antibiotic resistance gene intact
- origins of replication intact
- impacted reporter gene
plasmids are introduced into bacteria via
transformation
transformation uses two main techniques which are
- calcium ions and a heat shock to pulse DNA into cell
2. electroporation (high intensity pulse)
what is the blue-white screening mechanism?
- agar plates containing ampicillin
- agar plates contain x-gal
x-gal is blue when
bacterial cells with functional lacZ gene is carrying a nonrecombined plasmid
x-gal is white when
bacterial cells contain recombinant plasmid
what type of vectors are used to clone large fragments of DNA
BACs an YACs
the polymerase chain reaction is a
rapid method of DNA cloning
the polymerase chain reaction extends power
of recombinant DNA
PCR amplification requires r
- double stranded target DNA
- DNA polymerase
- Mg2+
- four deoxyribonucleoside triphosphates
- primers
- exponential increase of DNA molecule
the 3 steps of PCR are
- denaturation
- hybridization/annealing
- extension
a limitation of PCR is that it cannot
amplify long segments of DNA
what is known as the methodology for studying gene expression?
reverse transcription PCR
what is known as the method used to separate DNA fragments by size?
agarose gel electrophoresis
DNA sequencing has been through
computer-automated Sanger reaction-based technology
DNA sequencing enabled rapid progress of the
human genome project
what was discovered as a genome editing tool by scientists studying how bacteria fight viral infections?
CRISPR-Cas
CRISPR-Cas system uses
nuclease Cas9
CRISPR-Cas stands for
clustered regularly interspaced palendromic repeats
CRISPR-Cas takes advantage of
nonhomologous end-joining and nomology-directed repair mechanisms
with CRISPR-Cas cas9 or singleguideRNA are introduced into the cell to either
disrupt gene function or create nonfunctional alleles
