Topic 4 - Library screening and probes

Question 1

what is a probe (in the context of hybridization)

Answer 1

• relatively short ss fragment of DNA or RNA
• designed to be complementary to a specific target seq of RNA or DNA
• labelled with something that allows the target binding to be visualized!

Question 2

Q: Let’s say we wanted to create a mutagenized population of frogs using an insertion element to find frogs that didn’t jump normally. We used an insertion element to create the mutants (i.e., we inserted a piece of DNA into a gene to disrupt its function). What could you use to isolate the mutated gene by using a DNA probe?

a) We don’t have enough information to design a probe yet.
b) The wild-type gene that was mutated.
c) Frog genomic DNA.
d) The insertion element used to make the mutation.

Answer 2

d) use what we mutated it with!

Question 3

hybridization reactions can be done:
a) in solution
b) with target mixture bound to membrane
c) both
briefly explain what this would mean

Answer 3

c) both!
- solution ex. PCR (if primers were labelled)
- membrane ex. transfer denatured nucleotides to a membrane by capillary action (ss attached to membrane, membrane placed in a buffer -> labeled probe added in excess -> depending on incubation conditions (stringency) -> bind

Question 4

Q: Detection vs. isolation. Using a hybridization reaction, do we detect a specific region of DNA or isolate it?
a) Detect
b) Isolate
c) Both

Answer 4

a) detect
ex. but we can go back to the membrane and would know which colonies we were interested in

Question 5

two types of probes:
homologous/heterologous def

Answer 5

homologous - probe is exactly complementary to target seq
heterologous - probe for seq that isn’t perfectly complementary (e.g., gene family)

Question 6

distinctiveness for parts of gene:
what parts do we choose, for what scenario?

Answer 6

• ORF would be similar for similar genes (e.g., gene family)
• UTRs for very specific genes (e.g., NOT for gene families)

Question 7

when is DNA most stable (stringency)?

Answer 7

low temp
high salt
(low stringency)

Question 8

what is melting temp of DNA increased by? (3)

Answer 8

• more GC content
• higher salt conc
• longer DNA strands

Question 9

sensitivity of probe detection meaning?

Answer 9

very little probe bound -> still detectable

(want bound probe to be easily detectable and quantifiable)

Question 10

with what can you label a probe? (3)
how would you label probes (general)?

Answer 10

modifying nucleotide, adding it to probe somehow

• fluorescent molecule
  visualized with specific lights
• radioisotopes
  visualized using autoradiography
• molecule added to probe that is detectable w/ antibody (e.g., DIG)

Question 11

how we label the probes? (4)

Answer 11

• random priming
• PCR
• T4 polynucleotide kinase
• RNA probes (in vitro transcription; direction important)

Question 12

T/F: DIG-labelled probes (detected with antibodies) are stable for longer than radioisotope-labelled probes?

Answer 12

True!