Lecture 5: DNA and RNA manipulation

Question 1

what are the 3 diff kinds of blotting, and what are they for

Answer 1

• southern DNA-DNA
• northern DNA-RNA
• western antibody-protein

Question 2

what does a western blot detect

Answer 2

used to detect or idnetify specific proteins in homogenized tissue

Question 3

describe how southern blot works

Answer 3

1. a restriction enzyme is added to a solution containing the DNA of interest
2. the resulting fragments are separated by size by gel electrophoresis
3. after electrophoresis, the DNA fragment sin the gel are denatured and then transferred (blotted) onto filler paper
4. after blotting, the paper is removed
5. the paper is placed into a plastic bag w a solution w the single-stranded, labeled probe, the probe sticks to complementary frgaments by hybridization
6. to visualize the positions of the bands where the probe is hybridized, the filter paper is exposed to x-ray film, and either light or radioacative emissions darken the film over each band

Question 4

are DNA fragment bands visible during step 5 of southern blot

Answer 4

• no
• they need to be exposed to x-ray film and either light or radioactive emissions

Question 5

how does northern blot work

Answer 5

• Harvest total RNA
• Separated by agarose gel electrophoresis
• Hybridize a labelled probe to the blot
• Detect as before
• More RNA the band contains, the more the probe it will bind, and the darker the band will be on the film

Question 6

the more RNA the band contains in northern blllot, the ____ (lighter/darker) the band will be on the film

Answer 6

darker (cause itll bind to more probe)

Question 7

what are the target molecules in southern, northern, and western blot

Answer 7

• southern DNA
• northern RNA
• western protein

Question 8

how do you prep the samples in southern, northern, and western blot

Answer 8

• southern DNA extraction, enzymatic digestion
• northern RNA isolation
• western proteine extraction

Question 9

what are the separation methods for southern, northern, and western blot

Answer 9

• southern electrophoresis
• northern electrophoresis
• western electrophoresis

Question 10

what is the membrane material in southern, northern, and western blot

Answer 10

• southern nylon
• northern nylon
• western nitrocellulose or PVDF

Question 11

what probes are used in southern, northern, and western blot

Answer 11

• southern nucleic acid probe w sequence homologous to target
• northern RNA, DNA, or oligodeoxynucleotide
• western primary antibody

Question 12

what is the probe label in southern, northern, and western blot

Answer 12

• southern radiolabel, enzyme
• northern radiolabel, enzyme
• western enzyme

Question 13

what are the detection methods in southern, northern, and western blot

Answer 13

• southern x-ray film, chemiluminescence
• northern x-ray film, chemiluminescence
• western film, cooled CCD, camera, LED or infrared imaging system

Question 14

true/false southern, northern, and western blot all do separation by electrophoresis

Answer 14

true

Question 15

true/false southern, northern, and western blot all use x-ray film and chemiluminescence as a method of detection

Answer 15

• false
• southern and northern do
• western uses film, cooled CCD, camera, LED or infrared imaging system

Question 16

which 2 blot techniques have the same membrane material

Answer 16

• southern and northern
• nylon

Question 17

what are 2 ways to label DNA

Answer 17

• direct
• indirect

Question 18

true/false DNA interference is a defense mechanism

Answer 18

true

Question 19

what is the main purpose of RNA interference

Answer 19

to regulate gene expression

Question 20

What are the potential fates upon RNA interference in eukaryotes

Answer 20

• Rapid cleavage of target DNA
• Rapid translational repression, and (typically) eventual destruction of target RNA
• Formation of heterochromatin on DNA from which target RNA is being transcribed

Question 21

describe how RNA interference works

Answer 21

• Begins w the introduction of long Double stranded RNA (dsRNA). This can be natural (viral infections), or artificial (labs). it is formed vis complementary base pairing w itself
• This RNA is “cropped” while still in the nucleus and then exported into the cytosol
• in the cytosol, the enzyme “dicer” recognizes the dsRNA and cleaves it into short 20-25 nucleotide long fragments called siRNAs, or miRNA proper
• Argonaute, along with other components of RISC complex, associate with both strands of miRNA and then cleaves and discards one of them (the passenger strand)
• The other strand (guide strand) is loaded into the RISC complex
• RISC contains helices that unwind the siRNA, which can now bind to complementary mRNA sequences
• The guide strand binds to a complementary region on the target mRNA through base pairing, forming an RNA duplex
• If the RNA-RNA match is extensive, aeronaut cleaves the target mRNA causing rapid degradation
• If the RNA-RNA match is not longer than 7-nucleotide regions rapid inhibition of translation and eventual destruction will occur

Question 22

true/false in many organisms, the double-stranded RNA can trigger both the destruction of complementary mRNAs and transcriptional silencing

Answer 22

true

Question 23

can RNA interference direct heterochromatin formation

Answer 23

yes

Question 24

how can RNA interference direct heterochromatin formation

Answer 24

• The short siRNAs produced by the dicer protein are assembled with a group of proteins to form a RITS (RNA-induced transcriptional silencing) complex.
• This will bind complementary to RNA transcriptis as they emerge from a transcribing RNA polymerase
• The RITS complex will attract enzymes that covalently modify nearby histones and DNA causing the formation of a “constitutive” form of heterochromatin
• This will self propagate and prevent RNA from being transcribed

Question 25

true/false RNA interference is not a good way to test RNA function

Answer 25

• False
• RNA interference is a simple and rapid way to test gene function

Question 26

what are the steps of CRISPR

Answer 26

1) Short Viral DNA sequence is integrated into CRISPR locus
2) RNS is transcribed from CRISPR locus, processed and bound to Cas protein
3) Small cRNA in complex with Cas seeks out and destroys viral sequences

Question 27

what is CRISPR used for

Answer 27

gene editing

Question 28

what does CRISPR simplify

Answer 28

making transgenic organisms