wednsday week 3

Question 1

recombinant DNA

Answer 1

molecule that contains DNA from more than one source (ex. organism)

Question 2

tragenic

Answer 2

organism that contains DNA from different species

Question 3

what cuts DNA

Answer 3

restriction enzymes

Question 4

explain blunt vs sticky ends

Answer 4

blunt ends are when a restriction enzyme cuts a DNA sequence evenly so one could just put them back together, sticky ends are caused when restriction enzymes cut DNA in a non-even fashion so that one strand (top or bottom) is much longer than the other and they fit together like puzzle peices (as opposed to just lining up)

Question 5

DNA ligase

Answer 5

adds phosphodiester bond into sugar phosphorus backbone during the ‘pasting’ step of molecular cloning

Question 6

vector (plasmid)

Answer 6

small circular fragment of DNA, used to propagate DNA of interest in vivo

Question 7

3 features a plasmid MUST have

Answer 7

1. origin of replication
2. selectable marker
3. multiple cloning sites/polylinker

Question 8

step 1 of molecular cloning

Answer 8

cut and paste a sequence into the plasmid.

restriction enzyme cuts plasmid + DNA is cut with the same restriction enzyme so they can pair complementarily, DNA ligase is then added and joins fragments (gene of interest + plasmid)ste

Question 9

step 2 of molecular cloning

Answer 9

transformation.

incubate bacterial cells + DNA on ice then heat shock cells, and then plate under selection

Question 10

competent cells

Answer 10

cells with more permeable membranes to help bacterial cells take up the vector

Question 11

step 3 of molecular cloning

Answer 11

screen transformants for plasmids with insertions.

vectors with the DNA imbedded apear white on x-gal substrate (blue + white colonies)

Question 12

plasmid extraction (miniprep)

Answer 12

method used to harvest DNA grown in bacterial cells, the purified DNA can be used for other applicaitons (inject into tissue, sequence, etc)

Question 13

protein expression

Answer 13

method for making a bunch of protien (ex insulin) that we want/need

Question 14

reporter genes

Answer 14

method used to demonstrate which cells of an organism express the gene of interest - studies regulation (like electrohistochemistry for genes)

ex. cells glow florecent green, that carries the transgene that expresses green flourescent protien (GFP) from the promoter sequence of the gene of interest

Question 15

step 5 of molecular cloning

Answer 15

verify the DNA (content of vector, ex make sure it is not in upsidedown etc)

Question 16

restriction mapping

Answer 16

method used to map an unknown segment of DNA by breaking it into pieces and then identifying the locations of the breakpoint`

Question 17

items needed for sanger sequencing

Answer 17

1. labeled (radioactive) primer
2. template DNA to be sequenced
3. DNA polymerase
4. dNTP mixture
5. each tube gets a small amount of one of the four (ATP, GTP, CTP, TTP) ddNTPs

Question 18

ddNTP

Answer 18

dideoxynucleoside triphosphate (lacks 2’ and 3’ hydroxyl groups)

stops polypeptide additions because there is no 3’ OH end to bind to

Question 19

method for sanger sequencing

Answer 19

start with all of the required ingredients in four tubes (each gets either A, T, G, or C ddNTPs) – each tube will have a bunch of different sequences all of different lengths

use electrophloresis on the sequences in the tubes (one lane for each nucleotide, T, A, etc)

results:
original strand: read from top and read off the complementary base pair of the blot that shows up

synthesized strand: read directly off of electrophloresis blot from bottom up

Question 20

types of next generation sequencing

Answer 20

RNA sequencing and genome sequencing

Question 21

RNA sequencing

Answer 21

more effecient method of sanger sequencing

still sequencing DNA just starting with RNA. measures gene expression genome-wide

Question 22

genome sequencing

Answer 22

shotgun strategy (not good for complex, repetitive genes)

fast and not very accurate