L3 Flashcards
true or false, libraries can be constructed containing all the genes in an organism or all the genes expressed in a particular tissue
true
what kind of libraries do BAC vectors help construct
genomic libraries
what is a genomic library
a set of clones containing all of the genomic/ chromosomal DNA of an organism
what are the requirements for contructing a genomic library with a BAC vector
- DNA can be from any cell
- even representation of genome is crucial
describe the preparation of genomic library
- cut the DNA with blunt cutting RE, phosphotase (stops circularisation)
- prepare high MW DNA
- random shear, blunt ends
- gel purify large fragments
what is a cDNA library
set of clones containing DNA copies of all mRNAs expressed in a certain cell type of an organism
how are cDNA libraries constructed
- ) isolate population of mRNA from cells (oligo-dT column)
- ) anneal oligo-dT primer, copy to DNA with reverse transcriptase
- ) treat with RNAseH (attacks RNA moiety of RNA-DNA hybrid)
- ) treat with DNA pol 1 to make dsDNA
- ) clone into plasmids or lambda vectors
- can clone blunt, or ligate linkers
true or false, early cDNA clones often incomplete at 5’ ends, premature termination of RT, loss of bases at 5’ end during polishing, new method replace cap with a known oligo
true
Describe BAC genomic library
- source is chromosomal DNA
- all genes in an organism
- has introns, promoters, repeat sequences, TE (transposable elements)
- higher sequence complexity
- cloned as large fragments (in BACs, 100-150 kb)
- can be multiple (or partial) genes per clone
describe plasmid cDNA library
- source is mRNA
- genes expressed in source tissue
- has coding regions and adjacent transcribed sequences only
- lower complexity (<1.5% in human)
- small inserts (1-2 kb on average) in plasmid or lambda vectors
- always one gene per clone
true or false, to a large extent PCR amplification from either genomic DNA or cDNA has replaced construction and screening
true
what is the “catch 22”
- means that with PCR it requires that you know enough about your target gene so that you can design primers, however even today if you are looking for a totally new gene function a library screening approach is often still taken
true or false, making libraries is fairly straight forward, the real problem is finding the gene
true
what are the 3 common ways of identifying genes in a library
- probes
- antibodies
- complementation
how big is the mammalian genome
3 Million kb
what is the average BAC insert in a mammalian genome
100 kb
how large is a single coverage library for a mammalian genome
30 000 clones
how many clones does a representative library of a mammalian genome have
150 000 clones
what is the expected frequency for a single copy gene of in a mammal
1/30 000 BAC clones
true or false, although once you have identified the right BAC, the actual gene still needs to be subcloned
true
how large is the average human gene
14 kb
in a mammalian cell how much mRNAs
2 times 10^5 mRNAs
representative library of cDNA of mammal
one gene per clone, 1 million clones
true or false, in mammals the abundance of mRNA varies from 1 copy per 10 cells, up to 100 000 per cell
true
what is the expected frequency of cDNA library of mammalian cell
50% to 10-6
true or false, in mammalian cDNA library, the abundance can be zero if gene not expressed in the starting tissue; gene youre looking for must be expressed in the starting tissue
true
when do you use a cDNA library
- when looking for a coding region, source is mRNA, gene must be expressed in the source tissue
true or false, use of cDNA library allows expression of the genes in E.coli because there are no introns
true
true or false, the cDNA library needs to be constructed from a tissue where gene is highly expressed, if you don’t know this information, you need to use the genomic library
true
true or false, need to use a genomic library when the promoter, polyadenelation signal or introns are sought, rather than just the coding region
true
how to make a probe; method 1: colony or plaque colonisation using a labelled probe
the clone carrying the gene of interest is identified by probing a genomic library (looking for promoter). In this case made by cloning genes in a fosmid vector, with DNA or RNA known to be related to the desired gene.
A radioactive probe hybridises with any recombinant DNA incorporating a matching DNA sequence, and the position of the clone having the DNA is revealed by autoradiography
true or false, DNA can anneal if there is > 70% DNA sequence of homology over 100 bases
true
true or false, can adjust the stringency of hybridisation to get the kind of matches you need by changing the temperature ( or salt ).
true
true or false, annealing at 85 degrees in standard salt conditions will mean only perfect matches will stick
true
true or false, annealing at 65 degrees in standard salt means up to 20% mismatch will still anneal
true
true or false, we can manipulate stringency by manipulating salt concentration and temperature
true
how to design oligonucleotide probes from protein sequence
A short sequence of a protein is used to design a set of redundant oligonucleotides for use as a probe to recover the gene that encoded the protein, one of the set of probes will be a perfect match for the gene
- inserting the least degenerate 20 base region of codon combination, this must be present in the hybrid probe to ensure that it will stick to the target mRNA
Describe method 2: Antibody detection of an expressed protein
- purify protein
- inject purified protein into rabbit/mouse/chicken and purify the antibodies
- extract their blood
- construct an expression library (cDNA clones behind promoter, often as fusion protein)
- in lambdagt11- lambda insertion vector with lacZ gene- 1/6 of inserts express a fusion protein
- specificity resides in specificity of antibody binding
- use of secondary antibodies amplifies signal
describe method 3: Complementation of E.coli or yeast mutants
- has been widely used to clone conserved “core functions” such as genes involved in amino acid biosynthesis, transcription, translation, transport, cell division
- often needs mutation in either E.coli or yeast (and thus a selection for a functional gene)
- more difficult library construction- need to express complete functional copies of active proteins in E.coli or yeast, ie whole cDNA insert cloned into expression vector
