Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

BIOL 200 > PL4 > Flashcards

PL4 Flashcards

1
Q

describe steps (pathway) of recombinant DNA technology

A

vector + DNA fragment –> recombinant DNA –> replication of recombinant DNA within host –> isolation, sequencing and manipulation of purified DNA fragment

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

what is a vector

A

contents for replicating DNA derived from pieces of DNA that grows in bacteria

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

what is recombinant DNA

A

DNA from different sources
bacterial vector with some sort of eukaryotic DNA
want to produce more of that DNA for experiments

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

what are plasmids

A

most commonly used vectors
small
multiple copies per bacterial cell - aids replication

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

describe plasmids - 6

A

most common vector used in recombinant DNA technologies
circular
dsDNA (double stranded)
extrachromosomal (not in bacterial chromosome - exists separately)
found in bacteria and lower eukaryotes
Replication occurs before cell division (not linked to replication of chromosome, bacterial cells can have many copies of plasmids)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

what are restriction endonucleases or restriction enzymes

A

cleave or cut phosphodiester bones usually in symmetric fashion
Recognize sequence, bind and cut it

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

where do restriction endonucleases cut

A

site is symmetric - reads same 5’–>3’ (on both strands) = palindrome
Restriction enzymes recognize palindromes (sites)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

what are sticky ends

A

endonuclease makes staggered cuts at a specific sequence
ECORI = 4 base overhang
these can base pair to each other - only DNA cut with specific enzyme can anneal through sticky end with another piece cut with ECORI

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

a vector DNA cut with ECORI will only base pair with…

A

a genomic DNA fragment also cut with ECORI

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

what does a vector have

A

replication origin
Restriction enzyme site
some selectable marker

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

do all endonucleases cut directly at site they recognize

A

NO
some cut upstream - not useful fro DNA cloning since it will not be compatible for sticky ends

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

what are blunt ends

A

some restriction enzymes do not make staggered cut = they cut at same spot on both strands = blunt ends

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

are blunt ends used in DNA cloning

A

nO
SInce ligation reaction is not as efficient - since would not be helped by base pairing of sticky ends
maybe sometimes used for a specific reason

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

describe T4 DNA ligase

A

broken phosphodiester bonds are fixed by T4 DNA ligase
MAKES new bonds in phosphodiester backbone
seals recombinant DNA molecule

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

name the parts of engineering E coli plasmid

A

plasmid cloning vector with replication origin, gene that controls drug resistance (ampr) and polylinker

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

describe gene that controls drug resistance (engineered E coli plasmid)

A

want to be able to select bacterial cells that have this plasmid from ones that do not (if they took up drug resistance or not)
petri plating = cells with no plasmid will die on petri plate since plated on drug always

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

describe polylinker (engineered E coli plasmid)

A

area with many restriction enzymes
artificial sequence with many restriction enzymes all adjacent to each other - so can use same vector to clone DNA (cut with any of enzymes)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

how many restriction sites do we want for vector

A

1 site in vector
so it will cut then sticky ends have to anneal to fragment of DNA
do not want to cut vector into pieces (bad)

19
Q

is it useful to use single restriction enzyme or more than one

A

if use single enzyme then cannot control direction of how fragment inserted into vector - usually fine but if you have specific protein then wrong strand can be transcribed
if use 2 enzymes = cut bacteria with it - 2 ends, then cut vector with same - 2 sites, so each site will match with end of DNA cut by same restriction enzyme = can control orientation of fragment

20
Q

what is another advantage of using 2 restriction enzymes

A

sometimes there is noise because vector just ligates back to itself - if done with 2 enzymes then will not happen since sticky ends will be incompatible

21
Q

what makes up recombinant plasmid

A

plasmid vector and DNA fragment to be clone - cut with enzyme and produce recombinant plasmid

22
Q

describe transformation of recombinant plasmid

A

add DNA to cells and want them to slurp it up
mix E coli with plasmids in presence of CaCl2 and heat pulse = takes up DNA
also can use electroporation = shock so they take up DNA

23
Q

WHAT happens after transformation of recombinant plasmid

A

culture on nutrient agar plates with ampicillin = transformed cells survive and cells that do not take up plasmid die on ampicillin plates
plasmid replicates and cell multiplication = colony of cells each containing copies of the same recombinant plasmid

24
Q

what are DNA libraries

A

where permanent collections of genes can be obtained and maintained

25
Q

name and briefly explain 2 types of DNA libraries

A

genomic libraries = chromosomal DNA
cDNA libraries = represent mRNA present in a given sample

26
Q

describe how genomic library made

A

vector cut with BAMHI
4 base sticky end - 6 base recognition site, so 4^6 = BAMHI site comes up every 4096 nts
partially digest with Sau3A = much more frequent since 4 base recognition site (4^4=256) - produces same sticky ends as BAMHI (so compatible)
Sau3A = low concentration and cut small so only cut but not fully
then LIGATE

27
Q

describe how cDNA library made

A

uses reverse transcriptase (RT)
RNA –> cDNA (via reverse transcriptase)
RNA –> proteins (via translation)
producing complementary DNA strands from mRNA = complementary DNA (cDNA)

28
Q

describe generation and cloning of a cDNA copy of mRNA

A

10 steps
involves oligo-dc primers
make double stranded DNA
linkers for some short sequences that contain EcoRI can be added and gets cut with enzyme and cloned into vectors = have library

29
Q

what are uses of recombinant DNA construct (2)

A

where particular gene is expressed
- microarray and in situ hybridization techniques reveal mRNA expression, co regulation and localization
where particular mRNA might accumulate
- recombinant DNA expression vectors enable regulated expression of exogenous genes and production of proteins in prokaryotic and eukaryotic cells

30
Q

what does in situ hybridization reveal

A

spatial distribution of an RNA

31
Q

describe in situ hybridization

A

make clone of DNA and want to know which cells/tissues in embryo express it
Synthesize a probe that is labelled with biotin and denature probe and so iys single stranded and hybridize so it can get inside tissue
usually use cDNA since whole probe is complementary to mRNA (genomic DNA had introns)
sonic hedgehog mRNA in 10 day mouse embryo = gene tested
only seen in specific places

32
Q

what does microarray analysis do

A

Simultaneously measures levels of many mRNAs
allows you to do many hybridization experiments at same time

33
Q

describe microarray analysis

A

fibroblasts with and without serum
reverse transcribe to cDNA labeled with fluorescent dye (one colour per type (with/without serum))
mix together and then hybridize to solid support and that is how probes for thousands of different genes
microarray = short oligo peptides, order is known
colours = red (RNA after stimulation), green (RNA before stimulation), yellow (more or less same amount in both)

34
Q

what can cluster analysis identify

A

coordinately regulated genes
each column represents a different gene at times after addition of serum

35
Q

when is lac promoter active

A

in presence of lactose or molecule like lactose (IPTG)

36
Q

cloned genes can be expressed…

A

transiently or stably in cultured animal cells

37
Q

describe transient transfection

A

no selectable marker so eventually gets lost on cells
usually good enough for many experiments

38
Q

describe stable transfection

A

neomycin resistance
protein gets expressed and then integrated into host chromosome then continues to propagate and cell divides and plasmid replicates
must keep electing on plates with bacteria

39
Q

what can retroviral vectors be used to do

A

integrate cloned genes in mammalian genomes

40
Q

what is first step of using retroviral vectors to integrate cloned genes into mammalian genomes

A

construct virus that can infect certain cells of organism and integrate the DNA it carries into chromosomes of cells
by transient transfection of cultured cells with 3 different plasmids

41
Q

describe the 3 plasmids of using retroviral vectors to integrate cloned genes into mammalian genomes

A

vector plasmid = had 2 sequences derived from transposable elements (LTR - allows it to integrate into chromosome), must be with gene you want to express
packaging plasmid = carries many genes from virus that are needed to make structural components of virus
viral coat plasmid = encodes membrane proteins on surface of virus which determines type of cell virus can infect

42
Q

what happens when have all 3 components (using retroviral vectors to integrate cloned genes into mammalian genomes)

A

those with all 3 have all components to assemble virus inside them
so then virus is functional and it infects those cells and comes out of cells - so then virus can be purified

43
Q

what is a common virus used in using retroviral vectors to integrate cloned genes into mammalian genomes

A

lentivirus
powerful technology

BIOL 200 (29 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions