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Cell & Molecular Bio Exam 3 > Lec 16 Genetic Engineering > Flashcards

Lec 16 Genetic Engineering Flashcards

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1
Q

Molecular cloning
Restrictive enzyme what is it

A

AKA restriction endonuclease (RE)
found in ONLY bacteria and archaea providing a defense mechanism against invading viruses

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2
Q

Molecular cloning
Restrictive enzyme
what is restriction site

A

cut double stranded DNA at specific recognition sequences

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3
Q

Molecular cloning
restrictive enzyme
how many base pair cutter and what is an example

A

6-bp cutter
ex: EcoRI (1st enzyme isolated from E. coli Strain R)

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4
Q

Molecular cloning
restrictive enzyme
what is palindromic sequences

A

palindromic sequence
ex: GAATTC for EcoRI

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5
Q

Molecular cloning
restriction enzyme
what generates overhang

A

A zig-zag cut generates overhang (protruding) ends- by most Res

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6
Q

What joins DNA fragments in the presence of ATP

A

DNA Ligase

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7
Q

what are the three vectors

A

plasmid
viruses
artificial chromosomes

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8
Q

first vector plasmid
what is it

A

a plasmid is an extra chromosomal DNA molecule which is capable independently from the chromosomal DNA

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9
Q

Plasmid has Ori which is what

A

replication origin

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10
Q

Vector plasmid what is selectable marker example

A

ampicillin resistance gene

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11
Q

vectors plasmid what is LacZ

A

multiple restriction sites usually within a gene

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12
Q

what is vector artificial chromosomes

A

for larger insert and large-scale analysis of genomes

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13
Q

Molecular cloning (genetic engineering) what are the four steps

A

1) DNA cleavage
2) Ligation of gene into vector by DNA ligase in the presence of ATP. Now the recombinant plasmid is made
3) transformation
4) Screening

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14
Q

Molecular cloning (genetic engineering)
what is DNA cleavage

A

Both gene fragment & vector are digested by restriction enzyme

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15
Q

Molecular cloning (genetic engineering)
what is transformation in the steps

A

transformation- introducing the recombinant DNA into bacteria

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16
Q

Molecular cloning (genetic engineering)
what is screening

A

using selective medium containing antibiotic and substrate for lacZ enzyme called X-Gal

17
Q

Molecular cloning (genetic engineering)
Screening antibiotic ex
screening X-Gal what is it

A

antibiotic- ampicillin-clones that contain the plasmid will grow in the medium with ampicillin
X-Gal-clones that have the gene fragment inserted in the LacZ gene will turn white

18
Q

PCR stands for what and what is it

A

polymerase chain reaction and its using the concept of DNA replication

19
Q

PCR protocol

A

1) DNA template, gene specific primer pairs, free nucleotides & Taq DNA polymerase
2) Thermocycle program

20
Q

Thermocycle
94C

A

94C to denature the DNA template

21
Q

Thermocycle
55C

A

55C for primers to anneal to the template

22
Q

thermocycle
70C

A

for DNA synthesis (or primer extension)

23
Q

PCR does what

A

exponential production of specific DNA fragment

24
Q

Memorize Hae(3) and EcoR1 and Hind(3) and Not1

A
25
Q

memorize this

A
26
Q

what are plasmids, what are they used for and what are three essential components

A

small circular DNA
Used for cloning small pieces of DNA
3 essential components are
Origin of replication- allow independent replication
Selectable marker- allow presence of plasmid to be easily identified
Multiple cloning site (MCS) usually within a gene called LacZ, which is reporter gene

27
Q

what do artificial chromosomes allow for and what are two examples

A

Plasmids have limited insert size
Yeast artificial chromosomes (YACs)
Bacterial artificial chromosomes (BACs)
Allow for larger insert for large-scale analysis
of genomes

28
Q

explain first and second step in genetic engineering

A
29
Q

What is transformation

A

DNA uptake by bacteria

30
Q

what is transfection

A

DNA uptake by animal

31
Q

what is transduction

A

injection of DNA into host cells by virus

32
Q

what is conjugation

A

DNA transfer between two cells via direct contact

33
Q

explain third step of genetic engineering: transformation

A
34
Q

What are the standard steps in genetic engineering

A

step 1 cleavage with restriction nuclease
step 2 covalent linkage by DNA ligase
step 3 transformation
step 4 screening

35
Q

explain step four of genetic engineering: screening a and b

A
36
Q

Explain PCR
step 1 to step 3

A

Cell & Molecular Bio Exam 3 (6 decks)

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