L3

Question 1

true or false, libraries can be constructed containing all the genes in an organism or all the genes expressed in a particular tissue

Answer 1

true

Question 2

what kind of libraries do BAC vectors help construct

Answer 2

genomic libraries

Question 3

what is a genomic library

Answer 3

a set of clones containing all of the genomic/ chromosomal DNA of an organism

Question 4

what are the requirements for contructing a genomic library with a BAC vector

Answer 4

• DNA can be from any cell

- even representation of genome is crucial

Question 5

describe the preparation of genomic library

Answer 5

• cut the DNA with blunt cutting RE, phosphotase (stops circularisation)
• prepare high MW DNA
• random shear, blunt ends
• gel purify large fragments

Question 6

what is a cDNA library

Answer 6

set of clones containing DNA copies of all mRNAs expressed in a certain cell type of an organism

Question 7

how are cDNA libraries constructed

Answer 7

1. ) isolate population of mRNA from cells (oligo-dT column)
2. ) anneal oligo-dT primer, copy to DNA with reverse transcriptase
3. ) treat with RNAseH (attacks RNA moiety of RNA-DNA hybrid)
4. ) treat with DNA pol 1 to make dsDNA
5. ) clone into plasmids or lambda vectors
   - can clone blunt, or ligate linkers

Question 8

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

Answer 8

true

Question 9

Describe BAC genomic library

Answer 9

• 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

Question 10

describe plasmid cDNA library

Answer 10

• 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

Question 11

true or false, to a large extent PCR amplification from either genomic DNA or cDNA has replaced construction and screening

Answer 11

true

Question 12

what is the “catch 22”

Answer 12

• 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

Question 13

true or false, making libraries is fairly straight forward, the real problem is finding the gene

Answer 13

true

Question 14

what are the 3 common ways of identifying genes in a library

Answer 14

• probes
• antibodies
• complementation

Question 15

how big is the mammalian genome

Answer 15

3 Million kb

Question 16

what is the average BAC insert in a mammalian genome

Answer 16

100 kb

Question 17

how large is a single coverage library for a mammalian genome

Answer 17

30 000 clones

Question 18

how many clones does a representative library of a mammalian genome have

Answer 18

150 000 clones

Question 19

what is the expected frequency for a single copy gene of in a mammal

Answer 19

1/30 000 BAC clones

Question 20

true or false, although once you have identified the right BAC, the actual gene still needs to be subcloned

Answer 20

true

Question 21

how large is the average human gene

Answer 21

14 kb

Question 22

in a mammalian cell how much mRNAs

Answer 22

2 times 10^5 mRNAs

Question 23

representative library of cDNA of mammal

Answer 23

one gene per clone, 1 million clones

Question 24

true or false, in mammals the abundance of mRNA varies from 1 copy per 10 cells, up to 100 000 per cell

Answer 24

true

Question 25

what is the expected frequency of cDNA library of mammalian cell

Answer 25

50% to 10-6

Question 26

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

Answer 26

true

Question 27

when do you use a cDNA library

Answer 27

- when looking for a coding region, source is mRNA, gene must be expressed in the source tissue

Question 28

true or false, use of cDNA library allows expression of the genes in E.coli because there are no introns

Answer 28

true

Question 29

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

Answer 29

true

Question 30

true or false, need to use a genomic library when the promoter, polyadenelation signal or introns are sought, rather than just the coding region

Answer 30

true

Question 31

how to make a probe; method 1: colony or plaque colonisation using a labelled probe

Answer 31

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

Question 32

true or false, DNA can anneal if there is > 70% DNA sequence of homology over 100 bases

Answer 32

true

Question 33

true or false, can adjust the stringency of hybridisation to get the kind of matches you need by changing the temperature ( or salt ).

Answer 33

true

Question 34

true or false, annealing at 85 degrees in standard salt conditions will mean only perfect matches will stick

Answer 34

true

Question 35

true or false, annealing at 65 degrees in standard salt means up to 20% mismatch will still anneal

Answer 35

true

Question 36

true or false, we can manipulate stringency by manipulating salt concentration and temperature

Answer 36

true

Question 37

how to design oligonucleotide probes from protein sequence

Answer 37

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

Question 38

Describe method 2: Antibody detection of an expressed protein

Answer 38

- 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

Question 39

describe method 3: Complementation of E.coli or yeast mutants

Answer 39

- 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