PL4

Question 1

What is a vector in recombinant DNA technology

Answer 1

Piece of DNA able to propagate within a cell which you can insert DNA of interest

Question 2

Where can a vector grow

Answer 2

Bacterial or animal cell

Question 3

What is recombinant DNA

Answer 3

Fusing two DNA fragments that come from different sources

Question 4

What are the steps for recombinant DNA technology (overall)

Answer 4

Vector + DNA fragment
Recombinant DNA
Replication of recombinant DNA within host cells
Isolation, sequencing and manipulation of purified DNA fragment

Question 5

What does replication of recombinant DNA witching host cells mean

Answer 5

The DNA replicates itself with host cell’s replication

Question 6

What is the most common vector used in recombinant DNA technologies

Answer 6

Plasmids

Question 7

What shape are plasmids

Answer 7

Circular

Question 8

What kind of DNA is in plasmids

Answer 8

Double stranded dna

Question 9

What is the relationship between plasmids and chromosome replication and what does it mean

Answer 9

They are extrachromosomal

Plasmids don’t integrate into chromosomes (will not replicate with chromosome)

Question 10

Where are plasmids found

Answer 10

In bacteria and lower eukaryotes

Question 11

When does replication of plasmids occur

Answer 11

Before cell division

Question 12

What do restriction endonuclease s or restriction enzymes do

Answer 12

Cleave or cut phosphodiester bonds usually in a symmetrical fashion

Question 13

What kind of restriction sites do restriction enzymes usually recognize

Answer 13

Palindromic sequences

Question 14

What are sticky ends

Answer 14

The ends after a restriction enzyme has cleaved that are compatible with each other (complementary)

Question 15

Why is it that cutting out of recognition sites is not good (upstream or downstream of sequences)

Answer 15

Even if they give sticky ends, they are not compatible and not good for replication

Question 16

What is selectivity in restriction enzymes and sticky ends

Answer 16

Will only base pair with fragment cut with same restriction enzyme if ECORI or compatible ends

Question 17

What are the breaks in the phosphodiester backbone fixed with

Answer 17

DNA ligase

Question 18

How many restriction sites should plasmids have for the restriction enzyme used

Answer 18

One

Question 19

What does the polylinker have to isn’t present anywhere else in the plasmid

Answer 19

Sites for different restriction enzymes

Question 20

What are the three things a plasmid has

Answer 20

Polylinker, replication origin, and drug resistant site

Question 21

What is the ampr site of the plasmid show us

Answer 21

Helps us distinguish which cells are drug resistant and therefore which cells have taken up the plasmid

Question 22

What are the two parts in a recombinant plasmid

Answer 22

Plasmid vector and DNA fragment to be cloned that has been enzymatically inserted

Question 23

What is the process called where a recombinant plasmid gets put into a cell

Answer 23

Transformation

Question 24

What are the steps of transformation

Answer 24

Mix cells with plasmids in presence of CaCl2 and a heat pulse and culture on nutrient agar plates containing ampicillin

Question 25

Why does CaCL2 help with transformation

Answer 25

It breaks up the cell wall and makes bacteria cell more permeable to incoming plasmid

Question 26

Why is heat used to facilitate transformation

Answer 26

To go “whomph” and take up the DNA

Question 27

What is the replication of the plasmid not linked to

Answer 27

Cell division

Question 28

What part of the plasmid facilitates plasmid replication

Answer 28

Replication origin

Question 29

Why does the number of plasmids varies from cell to cell

Answer 29

Plasmid replication is not coupled to chromosome replication in cell division which is why number of plasmids is always greater than number of chromosomes (1)

Question 30

Where can permanent collections of genes be obtained and maintained

Answer 30

DNA libraries

Question 31

What are the two kinds of DNA libraries

Answer 31

Genomic libraries and CDNA libraries

Question 32

What do genomic libraries contain

Answer 32

Chromosomal DNA, represents as many genes (as much of genome) as possible

Question 33

What is the genomic DNA partially digested with and why in genomic libraries

Answer 33

Sau3A to be as random as possible to best get the entire genome and produces same ends as BamHI but comes across sites more often

Question 34

What do cDNA libraries represent

Answer 34

MRNA present in a given sample

Question 35

What happens to RNA when it undergoes reverse transcription

Answer 35

It becomes cDNA

Question 36

What will cDNA not contain

Answer 36

The entire genome

Question 37

What is generation and cloning of a cDNA copy of mRNA

Answer 37

Made DNA copies of the different mRNAs present being expressed in a given cell

Question 38

What uses could you make of the recombinant DNA construct

Answer 38

Microarray and in situ hibridization techniques Recombinant DNA expression vectors

Question 39

What do Microarray and in site hybridization techniques reveal

Answer 39

MRNA expression, co-regulation and localization

Question 40

What does recombinant DNA expression vectors enable

Answer 40

Regulated expression of exogenous genes and production of proteins in prokaryotic and eukaryotic cells

Question 41

What does in situ hibridization reveal specifically

Answer 41

Spatial distribution of an RNA

Question 42

When can you use in situ hybridization

Answer 42

If you have a recombinant DNA molecule that’s complementary to the RNA of a particular gene

Question 43

What does microarray analysis simultaneously measure

Answer 43

Levels of many mRNAs

Question 44

What are the two starting samples in microarray analysis

Answer 44

Fibroblasts without (non-growing cells) and with (growing cells) serum added

Question 45

What does each dot represent in a microarray analysis

Answer 45

A CDNA that represents a different gene in the DNA

Question 46

What happens if the spot is green in microarray analysis

Answer 46

Expression of that gene decreases in cells after serum addition (more mRNA before stimulation)

Question 47

What happens if the spot is red in microarray analysis

Answer 47

Expression of that gene increases in cells after serum addition (more mRNA after stimulation

Question 48

Is directionality in how you clone important

Answer 48

Yes, for transcription

Question 49

How can cloned genes be expressed in cultured animal cells

Answer 49

Transiently or stably

Question 50

What is the difference between transient transfection and stable stransfection

Answer 50

T: not making a stable cell line that maintains the plasmid, no selectable marker S: has marker, permanent

Question 51

What are the three retroviral vectors that can be used to integrate cloned genes into mammalian genomes

Answer 51

Vector plasmid, packaging plasmid and viral coat plasmid

Question 52

What is a genome

Answer 52

Usually DNA, the entirety of an organism’s hereditary information

Question 53

How can eukaryotic proteins be produced in E. coli

Answer 53

With the inductive lac promoter