Ch 18: Recombinant DNA and Biotechnology Flashcards
Restriction site
enzymes break bonds the specific sequence
palindromic DNA sequences
sequences read the same forward and backward
sticky ends
DNA that is cut unevenly (staggered cuts)
gel electrophoresis
a mixture of fragments in placed in a well in a semisolid gel and an electric field is applied across the gel
negatively charged NDA fragments move towards positive end
the smaller the DNA fragment, the more it moves down
restriction enzyme
cuts DNA at specific site
recombinant DNA
DNA made in the laboratory that is derived from at least two genetic sources
DNA ligase
catalyzes the joining of DNA fragments
Recombinant DNA technology can be used to make
clones aka identical copies of genes
requirements for a vector/host
autonomous replication in chosen host
selectable marker for host cells
unique restriction enzyme site the vector that is not detrimental for insertion
Plasmid
circular piece of DNA
replicate independently of the main chromosome
add extra genes to the host cell’s genome (metabolic enzymes, conjugation, antibiotic resistance)
Viruses
replicate independently
infected cells have a different phenotype
can be altered to attenuate some detrimental genes in the virus
can be altered to carry recombinant DNA into cells
viruses as vectors
bacteriophages or retroviruses
selectable markers
detecting whether or not the gene of interest has been incorporated into the DNA/host
reporter gene
a gene whose expression is easily observed
lacZ gene
codes for an enzyme that can convert the substrate X-Gal into a bright blue product
transformation
Recombinant DNA is cloned by inserting it into host cells
transfection
recombinant DNA is cloned by inserting it into animal host cells
what is commonly used as eukaryotic hosts?
yeasts
Electroporation
a short electric shock that creates temporary pores in membranes
DNA shoots to the + end and can enter cels
Polymerase Chain Reaction (PCR)
in vitro
used to make more copies of DNA seqeuces
Steps of PCR
1) DNA fragments are denatured by heating g single-stranded DNA
2) Primers (one complimentary to each strand), plus dNTPs, and DNA
polymerase are added g annealing and extension g new double-stranded
DNA
3) Repeat (heating-cooling-heating….)
initial problem with PCR was
its temperature requirements
key insight was the use of Taq Polymerase
Can we mix DNAs from different species (rDNA or recDNA)
yes
What DNA functions are needed to make more
(vectors)?
Replication, selection, insertion site
How do you get DNA into, and then find it, in a host that can make more?
Transformation
DNA fragments used for molecular cloning come from two sources
Genomic DNA and cDNA
both kinds can be manipulated by mutation as well (site-directed mutagenesis)
Genomic DNA
from chromatin in the nucleus
cDNA
copy DNA or complementary DNA
from reverse transcription of mRNA
genomic library
a collection of DNA fragments that comprise the genome of an organism
The DNA is cut into fragments by restriction enzymes, and each fragment is inserted into a vector, which is used to produce a colony of recombinant cells.
cDNA libraries
Represent the mRNA population from a given cell/tissue.
cDNA is produced by making a DNA copy of the mRNA
population using the RNA-directed
DNA polymerase called, reverse
transcriptase (RTCase).
snapshot of the transcription pattern of the cell
used to compare gene expression in different tissues at different stages of development
to provide the ORF for expressing the protein
expression vectors
sequences needed for
expression of a transgene in a host cell.
expression vectors for prokaryote host
a bacterial promoter, ribosome binding site, and a transcription termination signal
expression vectors for eukaryotic host
eukaryotic promoter/enhancer and terminator (poly-A addition signal/site)
other examples of expression vectors
Inducible promoters which respond to a specific signal
• Tissue-specific promoters expressed only in certain tissues at certain times
• Signal sequences—e.g., a signal to secrete the product to the extracellular medium
DNA microarray
provides a large array of
sequences for hybridization experiments.
Ti Plasmid
tumor inducing
used as a vector for plant cells
plasmid has a region called T
DNA, which inserts copies of itself
into chromosomes of infected
plants.
rDNA inserted into Ti plasmids, then used to transform Agrobacterium cells, can allow inserted DNA into the plant chromosomes with the T DNA. Whole plants can be regenerated with new functions
conventional breeding
many generations
only genes from same species can be used
biotechnology
one generation;
genes from any organism can be sued
