Micro Ch 12

Front 1

What do restriction enzymes do for prokaryotes?

Back 1

protect prokaryotes from foreign DNA

Front 2

What type of restriction enzyme is more useful for specific manipulation of DNA and why?

Back 2

type II because these restriction enzymes cut within the recognition sequences

Front 3

Describe the structure of most restriction enzymes

Back 3

most restriction enzymes are homodimeric proteins (two polypeptide subunits each of which recognizes and cuts the DNA on one of its two strand-> double stranded break)

Front 4

How does a cell protect itself from its own restriction enzymes?

Back 4

modification enzymes

Front 5

how do modification enzymes protect a cell from its own restriction enzymes?

Back 5

each restriction enzyme is partnered with a modification enzyme that recognize the same sequence.
-modification enzyme methylates the restriction sequence which prevents the restriction enzyme from binding

Front 6

In gel electrophoresis, what is the gel made of?

Back 6

agarose, a polysaccharide

Front 7

What stain is used to see DNA under UV light?

Back 7

ethidium bromide

Front 8

define a vector

Back 8

simple genetic element such as a plasmid or a virus used to replicate an isolated gene

Front 9

What is the goal of molecular cloning?

Back 9

the isolate copies of specific genes in pure form

Front 10

What are the three main steps of gene cloning?

Back 10

1. isolation and fragmentation of source DNA
2. inserting DNA into cloning vector
3. introduction of cloned DNA into host

Front 11

What is a DNA (gene) library

Back 11

it's a mixture of recombinant constructs; some cells contain the desired cloned genes and other cells contain other cloned genes from the same DNA

Front 12

What properties make plasmids good cloning vectors?

Back 12

1.small size
2.independent origin of replication
3.multiple copy number (several copies are present in the cell)
4. selectable markers (like antibiotic resistance genes)

Front 13

how are vectors usually transferred?

Back 13

electroporation and transformation

Front 14

what's a polylinker/multiple cloning site

Back 14

a segment of artificial DNA that has cut sites for many restriction enzymes

Front 15

Give 3 characteristics of pUC19

Back 15

1. ampicillin resistances (selective)
2.polylinker within lacZ gene
3.lacZ gene

Front 16

How is insertional inactivation useful for detecting the presence of foreign DNA?

Back 16

foreign DNA inactivates the lacZ gene, which makes the colonies appear white instead of blue

Front 17

Define sequencing

Back 17

determining the precise order of nucleotides in a DNA or RNA molecule

Front 18

Describe the Sanger method.

Back 18

-copy the SS DNA using DNAP
-DNAP uses deoxyribonucleoside triphosphates as substrates, adding them to a primer
-dideoxy sugar lacks a 3' hidroxyl and elongation of the chain stops

Front 19

define PCR (polymerase chain reaction)

Back 19

a method that produces multiple copies of DNA in vitro

Front 20

What are the 3 steps of PCR?

Back 20

1. denature
2. anneal
3. elongation

Front 21

Describe in detail PCR.

Back 21

1. DNA is denatured into single strands with heat and excess of primers is added
2. as the mixture cools, DNA anneals to primers
3. DNAP extends the primers using the original DNA as the template
4. the strands are heat separated again and allowed to hybridize with the complementary regions of newly synthesized DNA

Front 22

What bacterium did thermostable DNAP first come from?

Back 22

Thermus aquaticus

Front 23

does taq polymeras proofreading activity?

Back 23

no

Front 24

Which bacterium did Pfu polymerase come from?

Back 24

Pyrococcus furiosus

Front 25

Why is Pfu polymerase better than Taq polymerase?

Back 25

-more thermostable
-proofreading capabilities

Front 26

T/F: during each round of PCR, the amount of product doubles

Back 26

true

Front 27

Describe reverse transciptase PCR.

Back 27

-reverse transcriptase is used to make a complementary DNA copy of an RNA sample
-PCR is used to amplify the DNA
-used to monitor gene expression

Front 28

Give two examples of when Reverse Transcriptase is used

Back 28

-monitor gene expression in an environmental sample
-clone eukaryotic genes by amlifying the mRNAs...avoiding the need to remove intrones

Front 29

Describe Quantitative (Real Time) PCR

Back 29

fluorescent probes are added to the PCR mixture
-upon binding to DNA fluorescence increases

Front 30

What is the advantage of QPCR?

Back 30

quick

Front 31

Define Site-directed mutagenesis

Back 31

uses synthetic DNA and DNA cloning technology to introduce mutations into a gene in vivo and at precise locations

Front 32

Describe the basic procedure of site-directed mutagenesis

Back 32

1. synthesize oligonucleotide primer with the desired mutation
2. allow the oligonucleotide primer to basepair with the single strand of DNA containing the target gene
3. pairing will be complete except for the region of mismatch at the mutation
4. DNAP extends the synthetic oligonucleotide

Front 33

Define cassette mutagenesis

Back 33

- restriction sites are close together, intervening DNA fragments are excised and replaced by a synthetic DNA fragment

Front 34

Gene disruption is a type of _____ mutagenesis

Back 34

cassette mutagenesis; cassettes are inserted into the midde of a gene, disrupting the coding sequence

Front 35

Define Gene Fusion

Back 35

wen segments from two different genes are engineered into one construct

Front 36

What are the advantages and disadvantages of using E. coli as a host?

Back 36

A. Well developed genetics
A. many strains available
A. best known prokaryote
D. some pathogenic strains
D. periplasm traps proteins

Front 37

What are the advantages and disadvantages of using Bacillus subtilis as a host?

Back 37

A. easily transformed
A. nonpathogenic
A. naturally secretes proteins
A. endospore formation simplifies culture
D. genetically unstable
D. genetics less developed than E. coli

Front 38

What are the advantages and disadvantages of using Saccharomyces cerevisiae?

Back 38

1. well developed genetics
2. non pathogenic
3. can process mRNA and proteins
4. easy to grow

1. unstable plasmid
2. will not replicate most prokaryotic plasmids

Front 39

4 ways transfection can occur

Back 39

1. phagocytosis of DNA by host cell
2. microinjection
3. electroporation
4. gene gun

Front 40

define transfection

Back 40

introduction of DNA into mammalian cells