Exam 2 Molecular Techniques 1

Front 1

what are the 4 basic ideas of recombinant DNA technology?

Back 1

-isolation
-amplification
-characterization
-alteration = manipulation

Front 2

a replicon is also known as a ...

Back 2

vector

Front 3

DNA cloning = ... cloning

Back 3

molecular

Front 4

4 essential steps of cell-based cloning:
-... of recombinant DNA
-...
-... to produce numerous cell clones
-... of recombinant DNA clones

Back 4

formation
transformation
amplification
isolation

Front 5

Step 1: formation of recombinant DNA
-purified ...
-purified ...
-separately digest with a ...
-add the digested component together and ...
-result is ...

Back 5

DNA
vector
restriction endonuclease
ligate
recombinant DNA molecule

Front 6

Step 2: transformation
-... the recombinant DNA molecules into the bacterial cell
-Key step, as the cells normally take up only # recombinant DNA molecule
-... or recombinant molecule occurs

Back 6

introduce
1
replication

Front 7

Step 3: Amplification to produce numerous clones
-the individual clone colonies are separated out on a ... dish for growth
-these colonies are then individually picked into a ... step (homogeneity)

Back 7

nutrient agar
secondary amplification

Front 8

Step 4-
... of recombinant DNA clones

Back 8

isolation

Front 9

What will you need?
-Enzymes: ...(scissors), ...(glue), ... (for PCR)

-... (vehicles)

-... (source DNA from which you will create a genomic library, cDNA library or simply a clone)

Back 9

restriction enzymes
ligases
DNA polymerases

vectors

Host cells

Front 10

restriction endonucleases (or restriction enzymes)
-cut double stranded DNA at a very specific ...
-recognize specific ... sequences
-produces either overhangs (... ends) or ... ends
-cleave DNA at specific sequences regardless of their ...

Back 10

restriction site
palandromic
sticky, blunt
origin

Front 11

Any 2 fragments of DNA produced by the same restriction enzyme can be joined together because DNA ligase rejoins sticky or blunt ends by reforming the ... bond

Back 11

phosphodiester

Front 12

what can you use to separate DNA fragments?

Back 12

gel electrophoresis

Front 13

migration through the gel matrix in an electric field is influenced by what 4 factors?

Back 13

size
shape
charge
chemical composition of molecule

Front 14

DNA and RNA migrate towards the positive end (...)

Back 14

anode

Front 15

why separate by electrophoresis?
-visual difference in fragment ... difference
-... of DNA fragment
-further analysis by ...

Back 15

length
purification
Southern blotting

Front 16

what is an electrode from which electrons flow out of creating a positive charge?

Back 16

anode

Front 17

what is an electrode in which electrons flow into creating a negative charge?

Back 17

cathode

Front 18

vector-plasmids
-naturally occurring extrachromasomal elements that ... autonomously (at high numbers)
-high copy numbers (amplification)
-selection (stably confer antibiotic ... to host)

Back 18

replicate
resistence

Front 19

What is this?
-DNA 'vehicle" that is able to replicate and maintain itself within a host cell
-contains polylinkers with restriction sites not found elsewhere in the plasmid
-used to carry insert DNA (up to 5kb); confers similar properties to insert DNA
-selectable by some phenotype to identify host cells which carry this

Back 19

vector

Front 20

The larger the insert size, the ... the overall yield of insert

Back 20

lower

Front 21

After a restriction enzyme cleaves the vector at the unique restriction site, the DNA inserts with compatible ends to the restriction site and is ligated via ligase into the vector.
The result is ... or ... (hybrid between the vector and the insert (foreign) DNA)

Back 21

recombinant DNA
chimera

Front 22

The next step is to get the recombinant DNA into a host for ... (transformation)

Back 22

amplification

Front 23

what is the process in which there occurs an uptake of naked DNA from the environment by a bacterial cell?

Back 23

transformation

Front 24

The host is used to grow your hybrid or chimera in to ensure rapid ... and high ...

Back 24

growth
yields

Front 25

2 different types of amplification:
-... bacterium replicates
-... replicates within the host (up to 500 plasmids)

Back 25

host
plasmid

Front 26

What is the ability of a cell to take up DNA from the environment (temporary disruption of cell membrane)

-you can do this through 2 ways, ... (heat shock) or ...

Back 26

competence

chemical treatment
electrical stimulation

Front 27

selection
-transformed cells are ... selection
-recombinant cells are ... inactivation (visual selection)

Back 27

antibiotic
insertional activation

Front 28

what is this?
-selective, exponential amplification from a heterogeneous population of DNA
-rapid, easy, sensitive, robust

Back 28

polymerase chain reaction (PCR)

Front 29

downsides to PCR
-sequence information (no longer a problem-HGP
-fragments usually ...
-the longer the fragment, the less ...

Back 29

short
amplified

Front 30

requirements for PCR:
-DNA
-primers (in excess)
-deoxyribonucleotides (in excess)
-heat stable DNA polymerase (taq polymerase, which is lacks 3'-5' exonuclease activity and has... fidelity(error prone); and Pfu polymerase, which has 3'-5' exonuclease activity and ... fidelity (low error rates)

Back 30

low
high

Front 31

PCR has a cyclic series of what 3 successive reactions?
-... of DNA
-primer ...
-DNA ...

Back 31

denaturation
annealing
synthesis

Front 32

We ... a PCR product because it provides us with large quantities for proper analysis. The amount of DNA material produced from a PCR reaction is limited

Back 32

clone

Front 33

what type of cloning is this?
-Taq polymerase leaves a 3' A overhang on PCR products
-ligated into vectors that have 5'T overhangs

Back 33

T-A cloning

Front 34

what type of cloning is this?
-polishing enzymes, including the Pfu polymerase, create blunt ends

Back 34

bunt end cloning

Front 35

what is this?
base pairing between the probe and the target

Back 35

hybridization

Front 36

what is this?
-complex mixture of unlabeled nucleic acid molecules
-in molecular cloning refers to specific DNA fragments (amplify)

Back 36

Target DNA

Front 37

what is this?
-labeled nucleic acid (single-stranded of double-stranded)
-working form must be single-stranded for base-pairing to occur
-labeled with radioactivity or fluorescence

Back 37

probe

Front 38

how do you remember what blot does what?

Back 38

SNOW DROP
-southern --> DNA
-northern --> RNA
-western --> protein

Front 39

after size fractionation by gel electrophoresis, the DNA is denatured and transferred to a membrane for hybridization, this is ...

Back 39

southern blot

Front 40

what are the 5 steps to blotting?

Back 40

-restriction enzyme digestion
-gel electrophoresis
-transfer
-hybridization
-detection