Selection of specific genes Flashcards
what is a gene library or genome library
a set of recombinant clones that contains all the dna from a specific organism
what is a metagenome library
all of the dna from a sample or set of samples
what are mrna and cdna libraries
recombinant clones that contain all the cda or mrna from an organism
what are metatranscriptome library
a cdna library of all organisms in a sample or set
what is partial digestion and why is it useful
partial digestion is the incomplete digestion of a fragment to increase the likelihood of genes being preserved
what enzyme is commonly used to cut genome libraries and why
sau3A1 is commonly used as it has a 4 base recognition system, GATC
the use of re that produces compativle sticky ends
what processes are typically used in eukaryotic cloning
due to high levels of introns and long genomes, typically cdna is used as well as high capacity vectors
why are cdna libraries used
each clone contains a gene,
clones contain produced products
clones are relevant to expression levels
expressible in bacterial calls
what are the two forms of screening
indirect and direct screening
what are the requirements for direct screening
knowledge of a gene such as antibiotic, biosythetic, degradative genes
what are some methods of inderect selection, where you know the sequence
hybridisation or pcr, protein and antibody preparation, ligand binding
degredation or biosythesis
what instances would hybridisation be used in screening
whwn the gene confers no observable phenotype
what is needed for a dna probe
knowledge of the gene to produce a complimentrary strand, as well as labeled to allow detection
what are the two types of probes
sense and antisense probes
what regions may eukaryotic probes be designed to attach to
intron and exon regions
what might be used as a probe
the gene itself
a pcr product
a homologous gene of a different species
an oligio nucleotide
what are the two forms of labelling and some examples
radio labelling - 32p atp
non radioactive markers such as biotin, dig and a flurophore
what processes are used to probe label
using labelled primers or dntps
nick translation
end filling
random priming
kleinow fragments
how does colony blotting hybridisation function
first cells are lysed
then dna is bound to nitrocellulose or nylon using uv or temprature
labeled probe is added, then washed, allowing for specific identification of colonies
how does dig dutp function
by using specific antidig antibodies that are labelled such as alkaline phospotase
how does biotin labeling work
biotin utp inseted in probe is bound to by avidin, attached to a flourophore, avidin has a high affinity for biotin
what is involved in immunochemical screening
primary antibodies that are speciic as well as secondary labelled ones that are detectable
what are the purposes of processing cloned regions
sequence analysis
restriction mapping
verificatiob of encoding genes
gene expression
what is rflp and what is it used for
restriction fragment legnth polymorphism is a graphical description of fragments produced by digestion by restriction enxymes
can also confirm insert orientation
