Brainscape's Knowledge GenomeTM

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


See full index

70 genetics > L7 - molecular bio techniques - DNA cloning & vectors > Flashcards

L7 - molecular bio techniques - DNA cloning & vectors Flashcards

You may prefer our related Brainscape-certified flashcards:
Biology 101 flashcards
1
Q

what does the ability to isolate and manipulate DNA allow us to do? (6)

A
  1. isolate & modify & understand genes
  2. identify gene seq of mutant phenotype
  3. manufacture proteins & vaccines
  4. create transgenic organisms
  5. diagnose genetic diseases
  6. develop gene therapy
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

what is recombinant DNA technology

A

isolating regions of DNA and copying them

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

describe process of Recombinant DNA technology

A
  1. restriction enzymes cut DNA into fragments

2. copy DNA (eg cloning vectors - bacteria - self rep)

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

example of cloning vector

A

plasmid

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

how do R enzymes work?

A

recognise specific sequences and cuts both strands of sugar phosphate backbone

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

what part of the DNA do REs cut

A

sugar phosphate backbone

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

what are sticky ends?

A

after the cutting of DNA by REs a small region of single stand may be exposed

bases can easily be added here

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

what are blunt ends

A

cleavage of DNA where no single strand is exposed

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

importance of REs in bacteria?

A

cut up virus DNA as protection

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

how do bacteria prevent REs cutting up their own DNA

A

have methylase which adds methyl group to restriction site of its DNA to prevent RE cleavage

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

how many pieces does ECOR1 cut human genome

A

730,000

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

what is restriction mapping

A

creating a map of the positions that REs will cut DNA

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

how is restriction map made

A
  1. DNA cut with individual RE(first)
  2. separate DNA by size via gel electrophoresis
  3. compare each restriction fragments distance moved to markers
  4. pairs of RE then used to identify their relative sites
  5. repeat many times to make map
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

what does restriction mapping tell us

A

how many times an RE cuts DNA

the position they cut the DNA

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

what are restriction fragment length polymorphisms (RFLPs) and why are they useful

A

DNA sequence differs between individuals.

if a sequence differs in the restriction site the sizes and amount of restriction fragments will differ

allows differentiation between individuals - forensics etc

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

features of plasmids that make them useful cloning vectors

A
  1. contain ori which means they can replicate independent of chromosome
  2. contain antibiotic resistance genes to allow selective growth of bacteria with plasmid
  3. fast replication
17
Q

describe process of producing coning plasmid

A
  1. cut plasmid and DNA fragment with same RE so they have the same sticky ends
  2. add the two together with DNA ligase
  3. DNA ligase reforms sugar phos backbone
  4. forms recombinant DNA plasmid which can be inserted into bacteria
18
Q

what is multiple cloning sequence?

A

stretches of DNA put into the plasmid that can be recognised by many REs

19
Q

why is multiple cloning sequence useful

A

plasmid can be opened by many REs so many options to match RE needed to cut DNA

20
Q

why is a method needed to identify recombinant plasmids

A

the plasmid can just open and close again so recombinant ones need to be identified

21
Q

describe method to identify recombinant plasmid

A
  1. MCS placed in lacZ gene
  2. lacZ gene codes B galactosidase which acts on X-gal to produce blue bacteria
  3. inserted recombinant DNA will disrupt gene
  4. blue colonies dont contain recomb DNA
    white colonies do contain recomb DNA
22
Q

list uses of cloned DNA

A
  1. create genomic library
  2. see organisation of genome
  3. identify changes in genome associated with phenotype/disease
  4. genetically engineer organisms
23
Q

what is the genomic library?

A

huge library of all different DNA fragments of genome kept in plasmids

24
Q

list other cloning vectors apart from plasmids

A
  1. bacteriophage vectors
  2. cosmids
  3. artificial chromosomes
25
Q

what are bacteriophage vectors (and how big of a fragment can they hold)

A

vectors based on viruses that infect bacteria

25-30 kb

26
Q

what are cosmid vectors

and how big of a fragment can they hold

A

modified plasmids

30-40 kb

27
Q

what are artificial chromosome vectors

and how big of a fragment can they hold

A

plasmids that can clone large DNA fragments
can only produce a few copies
100kb - 2mb

28
Q

how big of a fragment can plasmids contain

A

20kb

29
Q

why can eukaryotic DNA not be cloned? what is done instead

A

because it contains introns which are large

mRNA is cloned as it contains no introns and then converted back to DNA (cDNA)

30
Q

what is cDNA

A

complimentary DNA

complimentary to isolated mRNA

31
Q

how is mRNA converted to cDNA

A

via reverse transcriptase which makes single complimentary strand

32
Q

what are cDNA libraries

A

complimentary DNA copies of the mRNA (and therefore genes) expressed only in tissue the mRNA was extracted from

33
Q

describe process of cDNA production

A
  1. mRNA isolated
  2. reverse transcriptase complimentary base pairs DNA to mRNA
  3. mRNA digested with RNaseH to separate strands
  4. complimentary base pairing to single stranded DNA (DNA pol & DNA ligase)
  5. double stranded DNA ligated into cloning vector
34
Q

what is difference between cDNA library and genomic DNA library

A
  • genomic DNA library - fragments of entire genome

- cDNA library - only copies of mRNA present in certain tissues (eg kidney cDNA library, liver cDNA library)

35
Q

what are expression plasmids

A

cloning vectors that allow gene expression in bacteria
contain genes that direct T&T
used to produce large amounts of protein eg insulin

36
Q

what are eukaryotic expression vectors

A

vectors that allow expression of cloned genes in eukaryotes

contain genes that direct T&T

37
Q

define transformation

A

process of plasmid being taken up into cells

38
Q

why may not all replicated plasmids contain the desired insert?

A

plasmid could religate with itself instead of insert

70 genetics (19 decks)

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