Cloning a gene

Question 1

How do you do cloning by complementation?

Answer 1

Transforming a DNA library hopefully containing a WT copy of a mutant gene into a mutant cell, then scoring the transformants for a restored phenotype and minipreping the plasmid out of the transformant with the restored phenotype

Question 2

If we don’t get a transformant with a restored phenotype from doing cloning by complementation, what are 3 possible reasons why?

Answer 2

1. Library was incomplete and you didn’t have YFG+ in it
2. The mutation was dominant
3. There was a successful transformant but it wasn’t expressed

Question 3

What is another name for cloning by complementation?

Answer 3

Plasmid rescue

Question 4

What are 3 possible reasons for seeing a restored YFG phenotype, but there is no plasmid containing YFG?

Answer 4

1. Overexpression of another gene suppressed the effects of the original mutation
2. Transvection
3. Original mutation reverted

Question 5

What are 3 controls you would use for cloning by complementation?

Answer 5

Empty vector, plasmid curing, make a deletion based on the recovered clone

Question 6

Why would you do empty vector transformation as a control for cloning by complementation?

Answer 6

Proves that the restored phenotype is actually because of the YFG sequence on the vector and not the backbone

Question 7

Why would you do plasmid curing as a control for cloning by complementation?

Answer 7

If losing the plasmid causes the mutant phenotype again, we can say it was YFG+ that is restoring the phenotype

Question 8

After doing cloning by complementation, how do you be extremely sure that it YFG is the mutant gene?

Answer 8

One step gene replacement. Proves thats the mutant gene if replacing YFG+ in the genome generates the same phenotype as the original mutation

Question 9

What do you need to know about YFG to do one step gene replacement?

Answer 9

Upstream and downstream sequences in the genome. Get those by sequencing the vector

Question 10

What is in the PCR template to do one step gene replacement?

Answer 10

A selectable marker that will replace YFG

Question 11

What are the PCR primers for one step gene replacement?

Answer 11

3’ ends will bind to the template, and the 5’ ends have sequences that are homologous to regions upstream and downstream of YFG in the genome

Question 12

How does one step gene replacement tell us for sure that we have the right gene cloned?

Answer 12

If we replace the suspect gene with something else and get the mutant phenotype, that shows it’s the gene

Question 13

How can we get the original mutation once we know our gene?

Answer 13

Gapped plasmid repair

Question 14

What is a gapped plasmid?

Answer 14

Most of the YFG sequences are cut out with a restriction enzyme, leaving a small region of homology

Question 15

What happens if we transform a gapped plasmid into the original mutant?

Answer 15

The cell machinery will repair the gap to look like the original mutation in the genome, then we can miniprep that back out and sequence our original mutation

Question 16

What organisms does cloning by complementation work well for?

Answer 16

Yeast. A little impractical to do with flies or mice

Question 17

What is the only thing we need to know about our gene if we are doing positional cloning?

Answer 17

Approximate position in the genome, so we do need to map it

Question 18

What are the 2 approaches to positional cloning? When would you use each one?

Answer 18

Chromosome walking and databases. You would use chromosome walking if working with an organism that doesn’t have much genomic info available, or use a database if there is genomic info

Question 19

How do you start a chromosome walk?

Answer 19

Need to have a clone of at least one of the mapping genes. Generate a probe from one end or the other end of that clone. Then use that probe on a DNA library, and it will bind to any A sequences but also any other clone with sequences that are complementary to the probe. Then sequence that other clone and make a new probe from the end sequences of that one. Then probe the DNA library with the new probe and etc until you reach the other mapping gene

Question 20

What do you do once you’ve walked to the other mapping gene in positional cloning?

Answer 20

Take all the clones that had a probe bind and transform mutant cells with them and look for a restored phenotype

Question 21

How do we do positional cloning if we are working with an organism that has extensive genomic info?

Answer 21

Plug our two mapping genes into a database and it will tell you which genes are between them. Then just do cloning by complementation with those genes until you find one that complements

Question 22

When is homology based cloning useful?

Answer 22

When we have genes from a different organism, especially if we have genes from taxonomically diverse groups

Question 23

What do we look for in the homologous genes to do homology based cloning?

Answer 23

Highly conserved amino acid sequences

Question 24

What do you do with the highly conserved amino acid sequences from homologous genes in homology based cloning?

Answer 24

Build a pool of degenerate primers

Question 25

How do you build degenerate primers?

Answer 25

Use the genetic code, since multiple codons can encode the same amino acid. You follow the AA sequence and replace the third nucleotide in the codon with different ones that still encode the same amino acid whenever there’s degeneracy

Question 26

When building degenerate primers, what does R, Y, and X mean?

Answer 26

R: any purine can go in that position
Y: any pyrimidine
X: any nucleotide

Question 27

How do you determine which degenerate primer is the right one?

Answer 27

Test annealing at different temps. The primer that binds at the highest temp is the right one, and you can do PCR with that

Question 28

When do we use mutation based cloning?

Answer 28

When the original mutation was generated with a biological mutagen

Question 29

How do we do mutation based cloning?

Answer 29

1. Isolate genomic DNA from the mutant
2. digest it with a restriction enzyme that only cuts once in the P-element
3. Carry out intramolecular ligation
4. Transform E. coli with the fragments
5. Miniprep the plasmid out and sequence it

Question 30

How do we make sure intramolecular ligation happens?

Answer 30

Have a very low DNA concentration

Question 31

What is the only fragment that could successfully transform E. coli in mutation based cloning?

Answer 31

The one containing Ori, a bacterial selectable marker, and the flanking YFG sequences

Question 32

How do we do inverse PCR?

Answer 32

1. Isolate genomic DNA
2. Cut with restriction enzyme that only cuts once in P-element
3. Do intramolecular recombination
4. Do PCR with transposon sequences as primers, with the 3’ ends facing away from each other
5. Circular, so the PCR product you get are the YFG sequences flanking the P-element

Question 33

What is enhancer trapping?

Answer 33

Insertion of a P-element containing a reporter gene and its promotor between YFG and its enhancer, which causes the reporter gene to have the expression pattern of YFG

Question 34

How do we do a microarray?

Answer 34

1. Extract all mRNA from both sources
2. Reverse transcribe the mRNA with fluorescently labelled dNTPs, a different colour for each source
3. Mix the differentially labeled cDNA
4. Hybridize the mix to a microarray for that organism
5. Visualize the fluorescence

Question 35

How do you do subtractive hybridization?

Answer 35

1. Extract mRNA from both sources
2. Reverse transcribe one of the two samples with fluorescent dNTPs
3. Mix the cDNA of the 2 samples
4. Add a nuclease that only cuts double stranded DNA, everything that didn’t bind to something sticks around
5. Extract the single stranded sequences and either use them as probes or sequence them