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BMD211 Human Molecular Biology > Week 6 > Flashcards

Week 6 Flashcards

1
Q

What is a genome

A

The complete genetic material present in a cell

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2
Q

What is the transcriptome

A

Sum of the total mRNA expressed from the genes

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3
Q

Proteome

A

The entire complement of protein produced and expressed by a cell, tissue or organism

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4
Q

functional assays

A

systemic experiments used to explain the function of a protein in a cellular pathway and gene activity

include - flow cytometry,

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5
Q

what are the two DNA cloning methods

A

restriction enzymes
cloning vectors

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6
Q

restriction enzymes

A

used to cleave DNA at specific sites

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7
Q

cloning vectors

A

bacterial plasmids or viruses
to carry inserted foreign fragments

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8
Q

what is DNA cloning technique

A

joining together sequences from different sources
also called recombinant DNA

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9
Q

what is genetic engineering

A

changing genetic content
additions and deletions will be inherited

includes artificial changes in the DNA content
becomes part of the genome

creates a transgenic organism

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10
Q

genetic engineering process

A
  1. vector and DNA fragment
  2. forms recombinant DNA
  3. recombinant DNA replicates within host cells
  4. isolation, sequencing and manipulation of purified DNA fragment
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11
Q

what are nucleases

A
  • hydrolyse an ester within a phosphodiester bond
  • can be dna or rna specific
    needed for specific reassembly of dna fragments
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12
Q

what are the nucleases

A

endonucleases
restriction endonucleases - type II endonucleases
isoschizomers
neoschizomers

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13
Q

what are endonucleases

A

cleave WITHIN a nucleic acid chain - dna or rna

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14
Q

what are exonucleases

A

cleave from either END of dna or rna

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15
Q

what are restriction endonucleases

A
  • are bacterial enzymes
  • needed for production of recombinant dna
  • can cleave double stranded dna at specific sites

example - type II endonucleases

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16
Q

what are bacterial enzymes

A

enzymes found naturally in bacteria

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17
Q

what are type II endonucleases

A

most commonly used restriction endonuclease
same recognition and cleavage site

different from type I as those sites are far from each other

specificity may vary - some might allow similar sequences

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18
Q

how can type II restriction endonucleases be cut

A

can be staggered with sticky ends
- single stranded complementary overlap

blunt double stranded cut
- no overlap
- can still be useful in labs

19
Q

what are isoschizomers

A

pairs of two different enzymes
both specific to the same recognition site
cut in the same way

20
Q

what are neoschizomers

A
  • different enzymes with the same recognition site
  • cut differently can create different sticky ends
21
Q

what is a modification enzyme

A

produced by bacteria for each restriction enzyme with the same recognition sequence - usually a DNA methyltransferase

as a methylated recognition sequence cannot be cleaved by the restriction enzyme
a bacteriums own genome is protected by modification

22
Q

what is a vector

A

cloning requires a specifically engineered vector
often as a plasmid
can be used to propagate an incorporated dna sequence in the host

23
Q

what are some features of vectors

A

ORI, selective marker, engineered multiple cloning site, dna ligase

24
Q

what is an ORI

A

origin of replication
dna sequence that signals the start

25
Q

what is a selective marker

A

identifies transformants to allow for growth
and eliminates non-transformants

26
Q

what are transformants

A

cells with foreign genetic material

27
Q

engineered multiple cloning site

A

a synthetically generated sequence of DNA
contains multiple restriction sites
series of tandem (two or more) restriction endonuclease sites

usd in cloning vectors to create recombinant molecules
different enzymes can be chosen to create specific sticky ends

28
Q

what is dna ligase

A

used for dna fragments with sticky or blunt ends

29
Q

How is bacterial transformation done in the lab

A

Done by calcium chloride
And heat shock

Not effective so selective marker is used to collect the recombinant dna that have acquired antibiotic resistance

30
Q

Why is bacterial amplification and purification done

A

To clear negative bacteria
And select the positive bacteria that contain the plasmid

31
Q

How is bacteria containing the plasmid amplified

A

Colonies are selected and grown in liquid broth containing the antibiotic
Negative bacteria dies out and positive bacteria survive and give rise to more colonies

32
Q

How is plasmid dna isolated from the bacteria

A

Alpine lysis based spin colomn

33
Q

How do false positives of plasmids occur

A

Plasmid recircularises without a cloned fragment

34
Q

What is blue white selection

A

Allows bacteria with a vector and insert to be identified

35
Q

How does blue white screening work

A

The multiple cloning site is in the LacZ operant
LacZ produced beta galactosidase which turns blue when X-gal is added
So plasmids that contain the dna fragment interrupt beta galactocidase activity - and appear white
The white colonies are selected

36
Q

What is a dna library

A

Collection of dna molecules that are cloned into a vector
Effectively represent all dna sequences in the genome

37
Q

Pros/cons of dna library

A

Useful for representing the genome of simple organisms
cDNA is more useful for higher eukaryotes

38
Q

What is cDNA

A

Synthetic ‘copy’ DNA
That has been transcribed from mRNA
- only has exons

39
Q

How is cDNA made

A
  • copies of mRNA are collected and synthesised into plasmid vectors

MRNA is easily collected by a primer for the polyA tail

Enzyme reverse transcriptase is used to synthesise a complementary DNA to each mRNA - makes a single stranded cDNA

Which is then converted into a double stranded molecule

40
Q

How is cDNA inserted into a vector

A

Sticky end or linked is needed
Dna pol synthesises a second strand

Fragments are protected by methylation at EcoRi sites

Synthetic linkers with EcoRI restriction site is added to double strand cDNA

41
Q

What are the limitations of cDNA

A

Needs to be transformed (put into) E.coli
Only represents expressed DNA - exons
Silent genes not represented
Over expressed genes will be overrepresneted

42
Q

what is screening the cDNA library

A

required to identify clones with the interested gene

43
Q

what is hybridisation

A

ability of complementary dna or rna molecule
to associate with each other by base pairing

can be used to identify specific nucleic acids - when it is of a labelled oligionucleotide

44
Q

what is a probe

A

a radioactive nucleic acid - dna or rna

used to identify a complementary fragment

and locate the DNA sequence

signal appears over plasmid dna that is complementary to the probe

BMD211 Human Molecular Biology (2 decks)

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