in one nucleotide of DNA what are the 3 major moieties (components) that make it up?
- 5 carbon sugar
- phosphate group
- nitrogen bases
what is
... [Show More] the overall charge of DNA and on which moiety is it found
DNA is overall negative, it is found on the oxygen of phosphate backbone
name a method that uses the fact that DNA is charged to get that method to work?
Agarose gel electroporesis
what types of bonds are formed during hybridization when a strand of DNA finds its complement
hybridization is the formation of hydrogen bonds
name 3 differences between DNA and RNA
1. on sugar, RNA has an OC on C-2' (ribose), and DNA has an H on C-2' (deoxyribose)
2. nitrogenous base: RNA uses Uracil in plase of thymine
3. RNA is typically single stranded and DNA is double
dNTPs
DNA building block
NTPs
RNA building block
ddNTPs
terminator for sequencing, not naturally occurring
Replication
building blocks: dNTPs
enzyme/cellular machine to add that building block: DNA polymerase
Transcription
building block: NTPs
enzyme/cellular machine to add that building block: RNA polymerase
Translation
building blocks: amino acids
enzyme/cellular machine to add that building block: ribosome
You work in a lab and you have been asked to do a DNA extraction from bacterial cells. in one of the first steps you notice that you need to add the enzyme lysozyme. what is the purpose of adding this?
it breaks down the peptidoglycan cell wall
if you were instead extracting DNA from human blood cells, would you need to add lysozyme? briefly state why or why not?
No because the human blood cells do not contain a peptidoglycan cell wall
what chemical is added to precipitate DNA at the end of DNA extraction protocol so it can be concentrated?
alcohol (ethanol) is added to precipitate DNA to prepare it to be concentrated.
After a DNA extraction you can check the success of your extraction by either a spectrophotometer reading or running an agarose gel. list one reason why a spectrophotometer reading is more commonly done
it is much faster and cheaper to run
under what conditions might you choose to run an agarose gel instead (hint: what can you learn from a gel that you dont get with a spec)?
agarose gel gives you more information about the actual makeup of DNA, you can see the band sizes and know the size of components based on how far they traveled. (check to see if its smeared when you want to see your DNA [?])
after DNA extraction you can perform a quick spectorphotometer reading at what wavelength do you measure the following
DNA__________ RNA _____________ contaminants_______________
dna: 260 nm
rna: 250 nm
contaminents: 280 nm
what concentrations of agarose should be used for optimal seperation of DNA fragments of 100, 250, and 350 bp in length?
a. 0.5%
b. 0.8%
c. 2.0%
d. 12%
c. 2.0%
small bands= large concentration
to monitor the progress of electrophoresis, which of the following is added to a sample that doesn not associate with the DNA and runs ahead of the smallest fragments in the sample
a. ethidium bromide
b. polyacrylamide
c. bromophenol blue
d. SYBR Green
c. bromophenol blue
tracking dye that doesnt bind with dna
a- florecent tag typically used for agarose electroporesis
b- polymer formed from acrylamide subunits
c- asymmetrical cyanine dye used as a nucleic acid stain, binds to DNA (used in PCR)
a PCR machine cycles between 3 temperatures "steps". what is the name of each step and what is occurring during that step
1- 94 degrees c
2- 55 degrees c
3- 72 degrees c
1- denaturation, melting of DNA to become single stranded
2- annealing, hybridization of primers
3- extension, polymerization of DNA
why might the temp in step 2 be changed in different PCR runs?
the temperature depends and is adjusted based on the Tm of the primers (melting temperature)
why does step 3 always occur at 72 degrees c?
72 degrees is the prime temperature that taq DNA polymerase can polymerize DNA, the extension temp never varies
what determines the length of time required for step 3?
the size of amplicon determines the length of time for extension
Name 2 applications of restriction enzymes
1. clonning
2. DNA fingerprinting (RLFP)
a linear DNA has two BamH1 recognition sites after digestion by BamH1 how many fragments were generated
a. 1
b. 2
c. 3
d. 4
c. 3
a circular DNA has two BamH1 recognition sites after digestion by BamH1 how many fragments were generated
a. 1
b. 2
c. 3
d. 4
b. 2
In PCR reaction it takes a few cycles to generate the correct amplicon size. in the first cycle are the products too long or too short
too long- because it is stopped by a temperature change back to the 94 degrees c and the end is not reached
if you want to detect the presence of multiple pathogens (eg. 3 total) in a single specimen what method would you choose
multiplex PCR
if you wanted to detect an RNA virus in a given specimen what method would you choose?
reverse transcriptase
List the reagents needed to set-up a real-time PCR reaction
-taq DNA polymerase
-buffer
-template DNA
-2 primers
-dNTPs
-fluorescent probe
state 2 additions to the standard PCR machine that make it a real-time PCR machine. these differences explain why it is more costly
-need additional computer software and database
- need additional optics to detect fluorescentce
draw an example graph of data from a real-time pcr run. lable both axis and show the ct value on graph
y axis- concentration of flourescence
x axis- PCR cycle #
threshold line is important
ct value-> value at wich it proceeds above the base line
the following refer to PFGE:
what is the purpose of this method
to detect large DNA fragments
its used in DNA fingerprinting and identifying foodborn pathogens
compare and contrast PFGE with standard gel electrophoresis
one similarity?
they both detect DNA band sized/ if run was successful
one difference?
PFGE detects very large dna band sizes while standard detects normal to smaller sizes
draw what the electrical field looks like in PFGE and diagram how it changes in time
It is a hexagon shape with + on one side and - on the other. the electrical field moves from - to + direction but doesnt have one swift motion, it bounces around between the different sides
what is the purpose of doing a restriction digestion?
it is used in cloning in order to cut DNA to get a different gene to express what one gene has already expressed
what method is always performed following a restriction digestion to check results
agarose gel electrophoresis [Show Less]