DNA libraries

Question 1

What is a DNA library?

Answer 1

A collection of clones, with different libraries representing different types of clones

Question 2

What is a genomic DNA library?

Answer 2

A collection of clones that collectively represents all DNA sequences of the original organism

Question 3

What is in a genomic DNA library?

Answer 3

Every single piece of genomic DNA from the organism is in at least one clone, regardless if said piece actually does something

Question 4

What sort of vector would you use in a DNA library?

Answer 4

Depends on whatever organism you’re putting it into

Question 5

Does the genomic DNA library need to be from the same species it’s being put into?

Answer 5

No, the library can be from a different species

Question 6

How do you make a genomic DNA library?

Answer 6

Break up the genome by using restriction enzymes, then ligate them into vectors. Grow the vectors in E coli on selective media

Question 7

What might happen if a mutant cell is transformed with a vector from a genomic DNA library?

Answer 7

Complementation. If the WT sequence is on the vector, then it will restore the WT phenotype

Question 8

What are 3 possible reasons for not seeing a restored phenotype after transforming mutant cells with a genomic DNA library? How do we fix that?

Answer 8

The library may be incomplete, and not every sequence made it into a vector. The restriction enzyme used may have also cut in the complementing sequence, so that sequence is in multiple clones. We can either try to prepare the library again with the same enzyme, or try a different enzyme. The third reason could be that there is no promotor to transcribe the complementing sequence, but we can get around that by adding a yeast promotor to the YCP right before the MCS

Question 9

Does it matter which tissue a genomic DNA library came from?

Answer 9

No, every cell from every tissue has the same genome

Question 10

What is a cDNA library?

Answer 10

Collection of clones collectively containing all expressed sequences of the source organism

Question 11

Does it matter which tissue a cDNA library came from?

Answer 11

Yes, different tissues will be expressing different genes

Question 12

What are 3 things that need to be considered when creating a cDNA library?

Answer 12

Temporal conditions (adult vs embryo)
Spatial conditions (which tissue)
Growth conditions (environment influences gene expression)

Question 13

How do you make a cDNA library?

Answer 13

1. Extract mRNA from the target tissue and add polyT primers, reverse transcriptase, and dNTPs.
2. Add terminal transferase, which will add non-template nucleotides and act as a template for the complementary strand.
3. Use alkali hydrolysis to get rid of mRNA.
4. Add DNA polymerase and dNTPs to synthesize the complementary strand to get double-stranded DNA that represents the starting mRNA

Question 14

How can we remove the polyA and polyT tails from the newly made cDNA? Do we have to?

Answer 14

S1 exonuclease in very controlled amounts, or it will eat our cDNA. Optional step

Question 15

What are the 2 ways to get our cDNA into a vector to make the library?

Answer 15

1. Blunt end ligate

2. Ligate adaptors to the ends of the cDNA and then sticky end ligate the cDNA into the vector

Question 16

What are adaptors? Why would we use them?

Answer 16

Short sequences with restriction enzyme cut sites that we ligate onto the ends of our cDNA. Doing this will let us have more efficient ligation into the vector since sticky ends are much easier to ligate together than blunt ends. We can also choose two different restriction enzyme sites and then do directional ligation

Question 17

What has to be on the vector backbone when doing a cDNA library?

Answer 17

A promotor. The cDNA is only the expressed coding sequence and won’t have any regulatory sequences

Question 18

What is a problem with using blunt end ligation?

Answer 18

Inefficient compared to sticky end ligation, so can get an incomplete library

Question 19

How many copies of each clone will be present in a genomic DNA library? cDNA library?

Answer 19

The genomic DNA library will have the same number of copies for everything, while the number of clones for a cDNA library will be directly related to its expression level