Gene Isolation &Manipulation

Question 0

What protects the host genome from being digested?

Answer 0

methylation of A or C

Question 1

Restriction enzymes…

Answer 1

…digest DNA at specific sequences

…many are palindromes

Question 2

Are larger restriction sites recognized more often or less often?

Answer 2

less often

Question 3

What is more efficient in terms of ligating together? Blunt ends or sticky ends

Answer 3

-Sticky ends are more efficient

Question 4

What does DNA ligase do?

Answer 4

• seals DNA molecules that have been ligated together
• forms phosphodiester bonds
• produces a covalent circular recombinant molecule

Question 5

A circular DNA with one restriction enzyme site is cut with a restriction enzyme. How many half sites does it produce?

Answer 5

• 2

- if 2 half sites are ligated together, the restriction enzyme has 2 sites

Question 6

What is in a ligation mix?

Answer 6

non-ligated linear DNA and ligated circular DNA

-the reaction is not 100% efficient so it contains both elements

Question 7

Explain how cells are transformed with DNA.

Answer 7

1. ligation mix is added to competent E.coli. cells that have been treated with CaCl2. CaCl2 makes holes in the bacterial cell wall which inreases the rate of DNA uptake
2. ligation mix enters the E.coli. cells and is replicated
   (the circular plasmid will replicate because they contain an ORI, non-ligated linear molecules do not replicate)

Question 8

What is a copy number?

Answer 8

the number of plasmids per cell

• can vary from 1-700
• the most reliable ones are about 10 kb

Question 9

Are plasmids that contain pMB1origin of replication integrated into BAC?

Answer 9

No

-have a high copy number

Question 10

pUC18

Answer 10

-has multiple cloning sites that are next to each other and are unique

Question 11

Explain how to get a blue or white colony using a blue/white screen.

Answer 11

• Blue: beta-gal cleaves the substrate X-gal
• White: DNA is cloned into a multiple cloning site so beta-gal cannot be made because the lacA-alpha gene has been disrupted

Question 12

Explain how a blue/white screen works.

Answer 12

• allows for selection of recombinant plasmids
• lactose is cleaved into its monosaccharides by beta-gal
• beta-gal has 2 subunits: lacZ alpha and lacZ omega
• lacZ alpha is deleted from the host genome

Question 13

What are YACs and BACs used for?

Answer 13

• cloning large pieces of DNA
• YAC: 200kb
• BAC: 100kb, ORF comes from F episome
  
  • F episome has a copy number of 1 (low)

Question 14

Describe the process of southern blotting.

Answer 14

1. DNA is separated by gel electrophoresis
2. transferred to a membrane as ssDNA
3. incubated with a probe. Probe H-bonds using complementary bp
4. nitrocellulose paper can be placed to be visualized by X-ray film (autoradiography)

Question 15

Describe northern blotting.

Answer 15

• RNA is placed on nitrocellulose paper , immobilizing it
• then hybridized to radioactive DNA or RNA probes
• determines time or tissue where gene is transcribed into mRNA

Question 16

How is In situ RNA hybridization done?

Answer 16

• can be done with digoxygenin labeled antisense RNA that hybridizes to sense mRNA
• Dig-UTP is incorporated into antisense probe
• Dig is detected by an antibody

Question 17

Describe the process of Western Immunoblot.

Answer 17

1. protein is separated by SDS-PAGE
2. transferred to nitrocellulose paper
3. Nitrocellulose is incubated with a primary antibody that recognizes the protein
4. then incubated w/ a secondary antibody that recognizes the primary antibody and has an enzyme attached to it
5. when substrate of the enzyme is converted, the location of the primary antibody can be observed

Question 18

Describe how dideoxy sanger DNA sequencing is done.

Answer 18

Dideoxy nucelotides are incorporated into a DNA strand and act as chain terminators because they do not have a 3’ OH.

• there are fluorescent labels for each ddNTP
• each reaction has regular dNTPs so that DNA synthesis is not immediately terminated
• when a ddNTP is added, DNA synthesis is terminated
• end up w/ a pool of extended mcs
• mcs are separated by electrophoresis
• fluorecent mcs go past detection window, emit fluorescence that is recorded on a chromatogram

Question 19

What is PCR?

Answer 19

• start with a template DNA and specific primers
• priers hybridize to each end of the gene
• need 2 primers (1 for each strand) & excess dNTPs

Question 20

What are the steps for a PCR reaction?

Answer 20

1. Denature template DNA and primers at 95 C
2. Anneal primers to ss template DNA at 55 C
3. Taq polymerase extends primers using excess dNTPs at 72 C

• repeats 30 times
• amplified DNA increases exponentially

Question 21

PCR benefits

Answer 21

• primers can be designed if a sequence is known from cDNA or genomic DNA
• cDNA is made by reverse transcriptase
• PCR products that are amplified by cDNA can reveal how a gene is spliced
• can determine RNA level

Question 22

Describe real-time qPCR

Answer 22

• used instead of northern blots
• RNA is copied into cDNA with reverse transcriptase
• cDNA pool will reflect amount of mRNAs
• cDNA pool is used as a template for PCR
• SYBR green is added to PCR reaction (fluoresces)
• fluorescence is measured in real time to track PCR progress
  
  • more abundant mRNA starts amplifying sooner
  • less abundant mRNA amplifies after more cycles

Question 23

Describe high throughput DNA sequencing.

Answer 23

-Solexa coupled with Illumina
-get diff. fragments with known sequences at each end
-cluster generation by bridge amplification in the flow cell
-sequence by synthesis
-Illumia depends on a reversible terminator, FL-nucleotide acts as a chain terminator
-2nd round of elongation
-fluorescent tag is removed
-Illumina reads 75-100 bp at a time but can do many reaction at once
-

Question 24

Explain Pacific Biosystems.

Answer 24

• PacBio
• single mcs real-time (SMRT) cells have tiny cells called ZMW where light can shine from the bottom
• DNA polymerase is at the bottom of the ZMW, single mcs template is in each well
• fluorescent is added and light is detected
• DNA elongates for about 10,000 bp
• get long reads
• useful for de novo sequencing