X Molecular Techniques1 Dcom Mfm Exam2

Front 1

vector means the same thing as …

Back 1

replicon

Front 2

DNA cloning means the same as …

Back 2

molecular cloning

Front 3

foreign DNA means the same as … or … DNA

Back 3

insert
target

Hint 3

None

Front 4

what are the 4 essential steps in cell-based cloning?

Back 4

1. Formation of recombinant DNA
2. Transformation
3. Amplification to produce numerous cell clones
4. Isolation of recombinant DNA clones

Hint 4

None

Front 5

how do you form a recombinant DNA?

Back 5

separately digest purified DNA and vector DNA and with restriction endonuclease and then ligate together

Front 6

describe step 2 - transformation:

Back 6

introduce recombinant DNA into bacterial cells … the cells only take up one recombinant DNA

Front 7

describe step 3 - amplification:

Back 7

the transformed bacterial cells are plated and then individual colonies are picked up and grown in nutrient media

Front 8

what is the 4th step in DNA cloning in bacterial cells?

Back 8

isolation of recombinant DNA clones

Front 9

… cleaves DNA at a recognition sequence

Back 9

restriction enzyme/endonuclease

Front 10

restriction enzyme recognition sequences have the properties of being …

Back 10

palindromic
usually 4-6 bp in length

Hint 10

None

Front 11

the frequency of restriction sites on a DNA molecule depends on the …

Back 11

length of the recognition site

Front 12

DNA cleavage by restriction enzymes can produce either … or … ends

Back 12

blunt
sticky

Hint 12

None

Front 13

what are the 2 properties that a cleaved DNA has due to the sequence being palindromic:

Back 13

cleaved ends are complimentary
two strands have same nucleoide sequence running 5' to 3'

Hint 13

None

Front 14

a. EcoRI
b. SmaI

Back 14

identify the restriction enzyme belonging to the identified recognition site:
a.
b.

Front 15

what is the function of DNA ligases?

Back 15

rejoins sticky or blunt ends by reforming the phosphodiester bond

Front 16

a restriction enzyme with a recognition site that is 4 bps long will cut every … bases

Back 16

4^4 (258)

Front 17

a restriction enzyme with a recognition site that is 6 bps long will cut every … bases

Back 17

4^6 (4096)

Front 18

… is used to separate digested DNA according to it's size

Back 18

gel electrophoresis

Front 19

migration through the gel matrix in an electric field is influenced by … and … and …

Back 19

size
charge
shape

Hint 19

None

Front 20

the overall charge of DNA is …

Back 20

negative

Front 21

larger DNA fragments will migrate (faster/slower) than smaller fragments in gel electrophoresis

Back 21

slower

Front 22

what is the base pair difference that gel electrophoresis can detect?

Back 22

1 bp

Front 23

positive
negative

Back 23

Anode: an electrode from which electrons flow out of create a … charge
Cathode: an electrode in which electrons flow into creating a ... charge

Hint 23

None

Front 24

vector DNA is also know as a …

Back 24

plasmid

Front 25

plasmids are extrachromosomal elements that:

Back 25

a. incorporate themselves in host DNA
b. replicate autonomously

Hint 25

None

Front 26

the larger the insert size of the DNA the (lower/higher) the overall yield

Back 26

lower

Front 27

the hybrid - the combination of the vector and the insert (foreign) DNA is also known as …

Back 27

recombinant DNA

Front 28

heat shock is a method used for … (used to temporarily disrupt the cell membrane of the host bacteria

Back 28

transformation

Front 29

competence is …

Back 29

the ability of a cell to take up DNA from the environment

Front 30

PCR stands for …

Back 30

polymerase chain reaction

Front 31

what are the 4 ingrediants for PCR?

Back 31

1. DNA sample to be amplified
2. complementary primars for 3' flanking regions of your target DNA (in excess)
3. Deoxyribonucleotides (in excess)
4. Heat-stable DNA polymerase

Hint 31

None

Front 32

which DNA polymerase lacks 3' to 5' exonuclease activity and has a low fidelity?

Back 32

taq (thermus aquaticus)

Front 33

which DNA polymerase has 3' to 5' exonuclease activity and has a high fidelity?

Back 33

pfu (pyrococcus furiosus)

Front 34

PCR has 3 series of successive reactions, what are they?

Back 34

1. denaturation of DNA
2. annealing of primer
3. DNA synthesis

Front 35

why would one want to clone a PCR product?

Back 35

the amount of DNA from PCR reaction is limited

Front 36

what is T-A cloning?

Back 36

taq leaves a 3' 'A' overhang in PCR products and the products can be ligated into vectors with 5' 'T' overhangs

Front 37

pfu creates (sticky/blunt) end clones

Back 37

blunt

Front 38

briefly describe a southern blot:

Back 38

DNA is electorphoresed and transferred to a membrane. DNA clone is then used as a hybridization probe against target DNA from gel

Front 39

briefly describe a northern blot:

Back 39

RNA is electorphoresed and transferred to a membrane. a hybridization probe is then used to detect target RNA.

Front 40

briefly describe a western blot:

Back 40

proteins are size fractionated on a polyacrylamide gel and then probed with an antibody

Front 41

a working form of a hybridization probe must be ... stranded and must be ...

Back 41

single
labeled (radioactive or fluorescence)

Front 42

put the following steps in order:
a. Separate DNA digests by electrophoresis
b. Expose membrane to x-ray film
c. Digest with restriction endonuclease
d. Transfer DNA from gel to membrane
e. Hybridize membrane with probe

Back 42

c, a, d, e, b