Analyzing mixtures of DNA, RNA, protein

Question 1

Southern blotting vs northern blotting vs western blotting

Answer 1

Southern: DNA detection
northern: RNA detection
western: protein detection

Question 2

different types of labels/probes for DNA/RNA/proteins

Answer 2

DNA/RNA need labeled hybridization PROBE

proteins need a labeled ANTIBODY

Question 3

homologous vs heterologous probes

Answer 3

homologous probe: probe and target seq 100% match

heterologous probe: probe and target seq not 100% match

Question 4

higher homology allows higher _____

Answer 4

stringency

Question 5

what is meant by controlling stringency?

Answer 5

hybridization conditions (temp, salt conc) change min homology needed for hybridization
- also affected by probe size and sequences

Question 6

___ temp, ___ salt = high stringency

Answer 6

high temp, low salt = high stringency

Question 7

higher similarity - higher stringency &
lower similarity - lower stringency

Answer 7

higher similarity - higher stringency:
- less chance for mismatch

lower similarity - lower stringency:
- higher chance for mismatch

Question 8

Southern blot (general function, what can it be used for)

Answer 8

(capital S)
- detects specific DNA fragments NOT RNA

can be used for:
- estimate # and position of gene copies in genome
- restriction mapping of DNA fragments
- detect cloned seq
- detect transgenes
- detect homologous seq in diff genomes
- detect repetitive seq

Question 9

Southern blotting mechanism steps

Answer 9

• digest DNA with REs
• gel electrophoresis on fragments
• soak gel in NaOH (changes pH -> denature)
• transfer blot gel to nitrocellulose or nylon (stack on paper)
• fragments on membrane in identical position to on gel
• hybridize membrane with labeled probe
• expose membrane to X-ray film, showing hybridized DNA fragments

Question 10

temporal control meaning

Answer 10

genes that expressed at a precise time during life cycle, AKA developmental regulation

Question 11

spatial control meaning

Answer 11

• gene expressed a specific tissue or cell type
• AKA tissue-specific expression

Question 12

northern blotting mechanism steps

Answer 12

• mRNA is transcribed
• total RNA (all 3 major groups) isolated from cells, electrophoresed
• like Southern blotting but NO denaturation (RNA alr ss)
  – transfer to membrane, hybridize w/ radioactive probe, x-ray film
• can determine steady-state level = abundance of specific mRNA at certain time/conditions (dep on transcription and degradation rate)

Question 13

In situ hybridization

Answer 13

• probe binds to complementary nucleic acids within cell or tissue (probes are same as in Southern and northern)

like Southern and northern but identifies:
- genes directly in chromosomes (FISH - fluorescence in situ hybridization)
- transcripts (mRNA) directly in cell or tissue (developmental studies)

Question 14

microarray

Answer 14

probe is unlabeled
target is labeled (cDNAs made from isolated mRNA)

• isolate total mRNA
• reverse transcribe to cDNA, label
• mix, hybridize to microarray
• wash
• measure green and red fluorescence over each spot

Question 15

SDS page and western blotting

Answer 15

SDS - negatively charged detergent, binds to hydrophobic protein regions -> helps protein unfolding!

• separate based on molecular weight (size) instead of charge
• PAGE = vertical, whereas agarose gels = horizontal

Question 16

western blotting mechanism steps

Answer 16

• SDS page, separated by size
• proteins are transferred to membrane - western blotting
• membrane incubated with specific antibody
• bound antibody detected by *secondary antibody (labeled) for visualization