Section 3 - Module 12/13 Flashcards
Why clone DNA in living cells?
1) before PCR, cloning was the only way to copy (amplify) DNA sequences
2) to make more DNA with high fidelity for further study or manipulation
3) to produce substances of scientific or commercial value from genes
4) to modify the genomes of plants or animals to introduce new, desired traits
Do living cells or PCR copy DNA with more accuracy?
Living cells
What does cloning require?
a vector
What is a commonly used cloning vector?
Plasmid
Why are plasmids common vectors?
stable, self-replicating molecule, circular DNA molecule, there is a origin of replication, unique retrusion sites, has selectable markers
What is pUC 19?
A commonly used bacterial vector
What makes up pUC 19?
portion of the lacZ+ gene, with a restriction site ‘linker’ that contains numerous unique restriction enzyme cut sites, and antibiotic resistance gene
what is the pUC 19 antibiotic resistance gene?
ampR (for amplicon)
What is meant my ‘unique’ restriction enzyme cut site?
sites found no where else on the plasmid
What is ‘competent’ bacteria?
E. coli made receptive to transformation by chemical or electrical treatment
What is the lacZ gene in the transforming bacteria?
lac Z-, meaning they lack the portion of lac Z gene that is present in the plasmid
What happens to bacteria with no plasmid (transforming bacteria & selecting for recombination)?
do not grow since they have no antibiotic resistance
What do bacteria with non-recombinant plasmid produce?
beta-galactosidase, resulting in blue colonies
What do bacteria with recombinant plasmid produce?
do not produce beta-galactosidase resulting in white colonies
What do bacterial expression vectors include?
operon and regulatory sequences (allows expression of genes in bacteria)
what is cDNA
combined growth hormone gene
What is a measure taken to reduce the risk of transgenic salmon interbreeding with wild salmon?
Farmed salmon will be triploid (triploid females are sterile)
What is Rhizobium radiobacter (formerly known as agrobacterium tumefaciens) do?
Naturally transforms the DNA of higher plants
What does Bacillus thuringiensis produce?
a protein, Bt toxin, lethal to many insects
Desirable features of Bt toxin?
Toxicity specific to some insects, not toxic to humans & other animals, biodegradable
What is routinely used to check success of experiments?
PCR
What is routinely used in combination with RE cleave or PCR to check the success of cloning/transformation experiments?
Gel electrophoresis
When was “Celera Genomics will describe
the first assembly of the human genetic
code from the whole genome shotgun
method” said?
during the 1st draft of human genome accouchement under president Bill Clinton said by Venter
What is issue with shotgun sequencing?
problems with repetitive DNA sequences
How to fix issue of repetitive DNA sequences and shotgun sequencing method?
long-read sequences (like nanopore)
What are the advantages of long reads?
They help with the assembly and alignment of short reads
Base calling
greater read depth gives more confidence a base is accurately
Resequencing
When additional copies of a genome are sequenced
What is the transcriptome?
sequences sorted in to different genes
Applications of draft of human genome?
1) genome assembly by shotgun sequencing
2) transcriptomics, gene expression analysis
3) identifying species (DNA barcoding; the inventory of animal life)
4) studying microbiomes
5) environmental DNA (eDNA)
What DNA is most widely used for identifying animal species?
mitochondrial DNA (mtDNA)
Why is next gen good?
Good for mixed up and degraded ancient DNA, also good for detecting genetic traces of ancient hybridization in modern humans.