Plant Biotechnology Methods Flashcards
Organism containing foreign DNA
Transgenic
Process of inserting DNA into genome of cells
Transformation
Use of isotope type: 3H
Most biological molecules
Use of isotope type: 14C
Most biological molecules
Use of isotope type: 35S
Proteins
Use of isotope type: 32P
Nucleic acids, Phosphoproteins
Beta particle detector: Meter that counts current pulses and calculates rate (cps or cpm); typically used to survey for workplace contamination
Geiger-Mueller Detector
Beta particle detector: radioactive particles or photons produce series of excited states in fluor, output of fluor proportional to energy of interacting particle or photon
Liquid scintillation counter (LSC)
Small dsDNA with internal RE site, blunt-end ligated to cDNA, RE digestion of linker for insertion into vector
Linkers
Linkers with preformed sticky ends
Adapters
Fragment of DNA complementary to Gene of Interest
Gene probe
Gene you have and want to see what interacts with it
Bait
Protein that interacts with bait
Prey
Homologous genes from another organism
Heterologous probes
Requires knowledge of DNA or protein sequence
Synthetic DNA probes
Requires knowledge of DNA sequence to design PCR primers
PCR
Which method of DNA labeling involves Klenow fragment of DNA polymerase?
Random primer labeling
A large protein fragment that is produced when DNAP1 from E. Coli is enzymatically cleaved, has lost 5’ to 3’ exonuclease activity
Klenow fragment
Which method of DNA labeling involves DNAP1 and labeled dNTPs ?
Nick translation labeling
Which method of DNA labeling involves attaching a reporter enzyme to DNA ?
Non-radioactive direct labeling
Which method of DNA labeling involves specific binding proteins, nucleotides, and a reporter enzyme?
Non-radioactive indirect labeling
Blotting technique used for DNA
Southern blotting
Blotting technique used for RNA
Northern blotting
Which cloning method of antibodies involves forming hydridomas ?
Monoclonal antibodies
Which cloning method of antibodies involves drawing blood from the host and recovering the serum ?
Polyclonal antibodies
A good primer for PCR is typically how long?
20-30 bp
Maintain and propagate cloned DNA
Cloning vectors
Four different types of vectors
Plasmid, Phage, Cosmid, Artificial chromosome
Extrachromosomal DNA in bacterial cells; carries genes for antibiotic resistance, conjugation, and metabolism of unusual substrates
Plasmid
ORI that replicates with cell cycle
Stringent ORI
ORI that replicates faster than cell cycle
Relaxed ORI
Hybrids of phage lambda and plasmids; infects cell like a phage, replicates like a plasmid
Cosmid
Large insert capacity cloning vectors
Artificial chromosome
Linear molecule, inserted into SUP4 (suppressor tRNA marker)
Yeast artificial chromosome
DNA sequence with regulatory elements to get desired expression in the transformed plants
Expression cassette
What are the components of an expression cassette ?
Gene of interest, promoter, protein localization signal (if necessary), 5’-UTR and 3’-UTR sequences, and a selectable marker
What are the ways of obtaining a gene of interest ?
Genomic library screens, cDNA library screens, PCR, gift from someone who has gene
What is the most commonly used constitutive primer?
35S Cauliflower Mosaic Virus promoter
What is a chimeric promoter?
cis element from plant inducible genes + 35S promoter
What are the 5 components of MS medium?
Major inorganic nutrients, minor inorganic nutrients, iron source, organic supplement, and carbon source
Required specifically for root growth, more than one compound can behave like this one, also required for callus culture, somatic embryo induction, and plantlet regeneration
Auxin
Required specifically for sheath growth, promotes cell division, also required for callus culture, sometimes somatic embryogenesis and plantlet regeneration
Cytokinins
Large class of compounds of related structures, GA3 sometimes used to promote growth
Gibberellins
Used to promote distinct developmental pathways
Abscisic acid
Unorganized growing and dividing mass of cells
Callus
Densely aggregated callus
Compact
Loosely associated, easily broke apart callus
Friable
Plant cells with cell walls removed
Protoplast
