Lecture 13: Biotechnology

Question 1

genetic engineering

Answer 1

in vitro techniques to alter genetic material from prokaryotes

Question 2

where does biobrick assembly occur?

Answer 2

test tube

Question 3

heterologous expression

Answer 3

expressing a gene in a different host

Question 4

PCR

Answer 4

rapid amplification in DNA sequence copies

Question 5

thermocycler

Answer 5

automated PCR machine

Question 6

Quantitative PCR

Answer 6

amplifies RNA after conversion to DNA

Question 7

PCR steps

Answer 7

1. denaturation
2. flanking/binding
3. add DNA polymerase
4. Heat & cool

Question 8

reverse transcription PCR

Answer 8

makes DNA from mRNA template

Question 9

what does reverse transcription PCR do?

Answer 9

produces intron free eukaryotic gene

Question 10

what end is the primer added to in PCR?

Answer 10

5’

Question 11

Gel electrophoresis

Answer 11

separates DNA based on size & charge

Question 12

what electrode do nucleic acids migrate to?

Answer 12

positive

Question 13

what size of molecules move faster in gel electrophoresis

Answer 13

small molecules

Question 14

what does ethidium bromide do in gel electrophoresis?

Answer 14

is used to visualize the DNA fragment bands

Question 15

will the same DNA cut with different restriction enzymes have the same or different banding patterns?

Answer 15

different

Question 16

nucleic acid hybridization

Answer 16

DNA / RNA from 2 diff sources form a hybrid double helix

Question 17

nucleic acid probe

Answer 17

segment of DNA strand that has a predetermined identity

Question 18

southern blot

Answer 18

DNA is in the gel & probe is RNA/DNA

Question 19

northern blot

Answer 19

RNA is in the gel & the probe is DNA/RNA

Question 20

synthetic DNA usage

Answer 20

used for primers, probes & site directed mutagenesis

Question 21

what is synthetic DNA made of

Answer 21

oligonucleotides of 100 bases

Question 22

molecular cloning

Answer 22

movement of a gene from original source to small/manipulable genetic element

Question 23

what does molecular cloning result in?

Answer 23

recombinant DNA

Question 24

recombinant DNA

Answer 24

molecule containing DNA from different sources

Question 25

what 4 enzymes are used for cloning?

Answer 25

1. restriction endonucleases 2. DNA ligase 3. reverse transcriptase 4. DNA polymerase

Question 26

what does DNA ligase join together

Answer 26

joins 5' phosphate & 3' OH of adjacent nucleotides

Question 27

DNA polymerase

Answer 27

5' to 3' activity

Question 28

what do restriction endonucleases do

Answer 28

cuts DNA

Question 29

where are restriction endonucleases more commonly found

Answer 29

prokaryotes

Question 30

Type II restriction endonucleases

Answer 30

cleaves DNA within sequence - most useful - palindrome

Question 31

Type I

Answer 31

cuts DNA randomly & not within sequence

Question 32

Type III

Answer 32

cleaves outside of sequences

Question 33

recombineering

Answer 33

allows foreign DNA to be inserted into vectors/chromosome

Question 34

what was recombineering originally developed in?

Answer 34

saccharomyces cerevisiae

Question 35

where do cloning vectors replicate

Answer 35

the host

Question 36

why are plasmids used as cloning vectors?

Answer 36

small size & they have selectable markers

Question 37

pUC19 uses

Answer 37

insertional inactivation

Question 38

insertional inactivation

Answer 38

lacZ is inactivated & B-galactosidase is not produced

Question 39

blue vs white colonies

Answer 39

blue: vector is not inserted with foreign DNA white: vector is inserted with foreign DNA

Question 40

PCR cloning vectors

Answer 40

plasmids specifically for cloning DNA products made by taq

Question 41

where are shuttle vectors replicated & maintained

Answer 41

replicate & be maintained in E. coli & yeast

Question 42

yeast artificial chromosomes (YACs)

Answer 42

chromosomes that clone directly into yeast

Question 43

properties of a good host for cloning

Answer 43

1. non-pathogenic 2. rapid growth

Question 44

electroporation

Answer 44

electric pulse is applied to form pores

Question 45

microinjection

Answer 45

gene gun inserts into cells

Question 46

expression vectors

Answer 46

vectors that control expression of genes

Question 47

why are eukaryotic genes that contain introns easy to isolate?

Answer 47

because of poly (A) tails

Question 48

fusion proteins

Answer 48

join target & carrier proteins

Question 49

site directed mutagenesis

Answer 49

introduces mutations at a precise location

Question 50

what can site directed mutagenesis be used for?

Answer 50

assess amino acids activity in a protein

Question 51

where does site directed mutagenesis occur?

Answer 51

in vitro

Question 52

how many base DNA oligonucleotides can be obtained through site directed mutagenesis

Answer 52

12 - 40

Question 53

cassette mutagenesis

Answer 53

synthetic fragments that can change base pair changes

Question 54

gene disruption

Answer 54

inserts cassettes in middle of a gene

Question 55

knockout mutation

Answer 55

loss of function of a gene in which cassette is inserted

Question 56

reporter gene

Answer 56

encodes protein easy to detect & assay

Question 57

example of a reporter gene

Answer 57

green fluorescent proteins

Question 58

operon fusions

Answer 58

signals are fused with transcriptional signals of another gene

Question 59

protein fusions

Answer 59

2 proteins are fused & share same transcriptional & translational start/stop

Question 60

what do protein fusions yield?

Answer 60

1 hybrid polypeptide

Question 61

genome editing

Answer 61

uses CRIPSR from streptococcus pyogenes to alter eukaryotic genomes