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
Haplotype
segments are regions of chromosomes that have not been broken up by recombination that are shared by multiple individuals
Bioinformatics
merging molecular biology with computer technology, applied in databases to store, share and obtain
