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BIOL 200 > PL4 > Flashcards

PL4 Flashcards

1
Q

What is a vector in recombinant DNA technology

A

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

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2
Q

Where can a vector grow

A

Bacterial or animal cell

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3
Q

What is recombinant DNA

A

Fusing two DNA fragments that come from different sources

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4
Q

What are the steps for recombinant DNA technology (overall)

A

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

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5
Q

What does replication of recombinant DNA witching host cells mean

A

The DNA replicates itself with host cell’s replication

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6
Q

What is the most common vector used in recombinant DNA technologies

A

Plasmids

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7
Q

What shape are plasmids

A

Circular

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8
Q

What kind of DNA is in plasmids

A

Double stranded dna

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9
Q

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

A

They are extrachromosomal

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

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10
Q

Where are plasmids found

A

In bacteria and lower eukaryotes

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11
Q

When does replication of plasmids occur

A

Before cell division

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12
Q

What do restriction endonuclease s or restriction enzymes do

A

Cleave or cut phosphodiester bonds usually in a symmetrical fashion

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13
Q

What kind of restriction sites do restriction enzymes usually recognize

A

Palindromic sequences

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14
Q

What are sticky ends

A

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

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15
Q

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

A

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

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16
Q

What is selectivity in restriction enzymes and sticky ends

A

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

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17
Q

What are the breaks in the phosphodiester backbone fixed with

A

DNA ligase

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18
Q

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

A

One

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19
Q

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

A

Sites for different restriction enzymes

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20
Q

What are the three things a plasmid has

A

Polylinker, replication origin, and drug resistant site

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21
Q

What is the ampr site of the plasmid show us

A

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

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22
Q

What are the two parts in a recombinant plasmid

A

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

23
Q

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

A

Transformation

24
Q

What are the steps of transformation

A

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

25
Q

Why does CaCL2 help with transformation

A

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

26
Q

Why is heat used to facilitate transformation

A

To go “whomph” and take up the DNA

27
Q

What is the replication of the plasmid not linked to

A

Cell division

28
Q

What part of the plasmid facilitates plasmid replication

A

Replication origin

29
Q

Why does the number of plasmids varies from cell to cell

A

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)

30
Q

Where can permanent collections of genes be obtained and maintained

A

DNA libraries

31
Q

What are the two kinds of DNA libraries

A

Genomic libraries and CDNA libraries

32
Q

What do genomic libraries contain

A

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

33
Q

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

A

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

34
Q

What do cDNA libraries represent

A

MRNA present in a given sample

35
Q

What happens to RNA when it undergoes reverse transcription

A

It becomes cDNA

36
Q

What will cDNA not contain

A

The entire genome

37
Q

What is generation and cloning of a cDNA copy of mRNA

A

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

38
Q

What uses could you make of the recombinant DNA construct

A

Microarray and in situ hibridization techniques
Recombinant DNA expression vectors

39
Q

What do Microarray and in site hybridization techniques reveal

A

MRNA expression, co-regulation and localization

40
Q

What does recombinant DNA expression vectors enable

A

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

41
Q

What does in situ hibridization reveal specifically

A

Spatial distribution of an RNA

42
Q

When can you use in situ hybridization

A

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

43
Q

What does microarray analysis simultaneously measure

A

Levels of many mRNAs

44
Q

What are the two starting samples in microarray analysis

A

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

45
Q

What does each dot represent in a microarray analysis

A

A CDNA that represents a different gene in the DNA

46
Q

What happens if the spot is green in microarray analysis

A

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

47
Q

What happens if the spot is red in microarray analysis

A

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

48
Q

Is directionality in how you clone important

A

Yes, for transcription

49
Q

How can cloned genes be expressed in cultured animal cells

A

Transiently or stably

50
Q

What is the difference between transient transfection and stable stransfection

A

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

S: has marker, permanent

51
Q

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

A

Vector plasmid, packaging plasmid and viral coat plasmid

52
Q

What is a genome

A

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

53
Q

How can eukaryotic proteins be produced in E. coli

A

With the inductive lac promoter

BIOL 200 (13 decks)

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