Chapter 3 - Recombinant DNA Technology and Genomics Flashcards
Clone
a molecule, cell or organism that was produced from another single entity
Restriction Enzymes
DNA cutting enzymes
Plasmid DNA vectors
Circular form of self-replicating DNA
Restriction Enzyme process
Cuts doulbe-stranded DNA at its particular recognition sequence, making sticky ends’
Fragments join together. ligase unites the two backbones, making 1 molecule of recombinant DNA with both human and plasmid DNA
Selection of recombinant bacteria
some kind of marker needs to be added for selection
What makes a good vector
size, ori site, MCS, selectable marker genes, RNA polymerase promoter sequences , DNA sequencing primers
how do you select a vector?
based on insert size and use limitations
How do you identify and clone a gene of interest
Create DNA libraries. Two types:
genomic
complementary DNA libraries
Disadvantages of genomic libraries
Introns are clones in addition to exons
searching for a gene of interest is difficult and time consuming
cDNA libraries
advantages - introns not cloned, can isolate genes that are primarily expressed only under certain conditions
disadvantages - can be difficult to make if a source tissues with abundant mRNA is not available.
How do you identify and clone a gene of interest
PCR - add target DNA, nucleotides, buffer, polymerase add forward and reverse primers, put in thermocycler
each cycle consists of three stages - denaturation, annealing, extension
Northern blot
analyses mRNA. Studies gene expression
Genomics
cloning, sequencing, and analyzing entire geomes
Comparative Genomics
mapping and sequencing genomes from a number of model organisms
Whole genome shotgun
genome assemblies of incomplete
