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Biochemistry > Chapter 9: DNA-Based Information Technologies > Flashcards

Chapter 9: DNA-Based Information Technologies Flashcards

1
Q

genome

A

the complete haploid genetic complement of an organism

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2
Q

genomics

A

the study of DNA on a cellular scale (contributes to systems biology)

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3
Q

clone

A

an identical copy

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4
Q

DNA cloning

A

selective amplification of a particular gene or DNA segment so that its genetic information may be studied and utilized

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5
Q

recombinant DNA technology or genetic engineering

A

the methods used to accomplish DNA cloning and related tasks

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6
Q

the process of DNA cloning involves…

A

generating a recombinant vector

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7
Q

cloning vectors

A

small DNAs capable of autonomous replication

based off of plasmids- able to replicate themselves and propagate
example: bacteria, covid

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8
Q

recombinant DNAs

A

composite DNA molecules comprised of covalently linked segments form 2+ sources

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9
Q

advantages of cloning in E.coli

A

-DNA metabolism is well understood
-many naturally occurring cloning vectors (plasmids and bacteriophages)
-techniques for moving DNA from one bacterial cell to another

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10
Q

What enzymes are used to yield recombinant DNA

A

restriction endonucleases
DNA ligases

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11
Q

restriction endonucleases aka

A

restriction enzymes

recognize and cleave DNA at specific sequences (recognition sequences or restriction sites)

cleave phosphodiester bond

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12
Q

methylases

A

catalyze methylation of host DNA to protect it from digestion by the host cell’s restriction endonucleases

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13
Q

restriction-modification system

A

the restriction endonuclease and the corresponding methylase

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14
Q

dna ligases

A

joins the DNA fragment to be cloned to a suitable cloning vector

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15
Q

types I and III restriction endonucleases

A

large, multisubunit complexes containing both endonuclease and methylase activities

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16
Q

type II restriction endonucleases

A

simpler than types I and III
require no ATP
catalyze the hydrolytic cleavage of DNA Phosphodiester bonds within the recognition sequence

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17
Q

restriction sequences for some type II endonucleases

A

normally cleave at a specific site

typically 4-6 bp long
palindromic
cleave same spot on each side

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18
Q

sticky ends

A

unpaired bases on the ends
-due to endonuclases making staggered cuts
-can base pair with each other or complementary sticky ends

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19
Q

blunt ends

A

no unpaired bases on the ends
-due to endonucleases making straight cuts

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20
Q

the DNA Segment to be cloned is generated by

A

PCR

PCR is used to add the restriction site
-including restriction endonuclease cleavage sites facilitates the subsequent cloning of amplified DNA

(2 different restriction enzymes used- to permit proper orientation of DNA insert to plasmid, prevents ligation)

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21
Q

cleavage of PCR-amplified DNA creates

A

sticky ends used to ligate the amplified DNA to a cloning vector

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22
Q

linkers

A

synthetic DNA fragments created to bridge ligated ends

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23
Q

multiple cloning site (MCS)

A

inserted DNA fragment with multiple recognition sequences for restriction endonucleases
-useful for inserting additional DNA at a later point

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24
Q

three popular cloning vectors:

A
  1. plasmids
  2. bacterial artificial chromosomes
  3. yeast artificial chromosomes
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25
Q

plasmid

A

circular DNA molecule that replicates separately from the host chromosome

usually have symbiotic role in cell

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26
Q

key features of E.coli plasmid:

A
  1. origin of replication (ori)= sequence where replication is initiated
  2. resistance genes
  3. recognition sequences for restriction endonucleases
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27
Q

transformation

A

laboratory process by which small plasmids are introduced into bacterial cells through heat shock treatment
-becomes less successful as plasmid size increases

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28
Q

electroporation

A

laboratory process by which small plasmids are introduced into bacterial cells through high-voltage pulses
-transiently renders the bacterial membrane permeable

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29
Q

selectable markers and screenable markers are used to

A

identify cells that take up plasmid DNA

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30
Q

selectable marker

A

either permits the growth of a cell (positive selection) or kills the cell (negative selection) under defined conditions

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31
Q

screenable marker

A

gene encoding a protein that causes the cell to produce a colored or fluorescent molecule (visual change)

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32
Q

bacterial artificial chromosomes

A

plasmid vectors developed to allow the cloning of very long segments of DNA

BACs= composed of the plasmid vector and large segments of cloned DNA

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33
Q

the BAC vector

A

-have stable origins of replications that maintain low copy numbers
-contain par genes from an F plasmid that direct the reliable distribution of the recombinant chromosomes at cell division

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34
Q

CamR

A

positive selection marker in the bac vector

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35
Q

lacZ

A

screenable marker in the bac vector

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36
Q

yeast artificial chromosomes

A

YACS= composed of the plasmid vector and large segments of cloned DNA
*ori + centromere + 2 telomeres + selectable markers X and Y

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37
Q

DNA cloned in a YAC can be altered to study

A

-the function of specialized sequences in chromosomes metabolism
-mechanisms of gene regulation and expression

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38
Q

shuttle vectors

A

plasmids that can be propagated in cells of 2+ species
-incorporate multiple replication origins or other elements

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39
Q

the YAC vector

A

contains elements to maintain a eukaryotic chromosome in the yeast nucleus:
-yeast origin of replication (ori)
-two selectable markers
-specialized sequences for stability and proper chromosome segregation at cell division
–centromere (CEN)
–two telomeres (TEL)

*for bacteria, large areas of DNA are hard to keep but in yeast, larger you get, the more stable you become

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40
Q

pulsed field gel electrophoresis

A

segregates genomic fragments following partial digestion with restriction endonucleases
-used to obtain a suitable fragment size

41
Q

the stability of YAC clones

A

increases with the length of the cloned DNA segment (up to a point)
-inserts > 150,000 are very stable
-inserts < 100,000 bps are gradually lost during mitosis

42
Q

YACS that lack a telomere at either end are

A

rapidly degraded

43
Q

expressing a eukaryotic protein in a bacterium

A

eukaryotic genes have surrounding sequences needed for their transcription and regulation
-sequences do not function in bacteria

44
Q

expression vectors

A

cloning vectors with transcription and translation signals needed for the regulated expression of a cloned gene

45
Q

in principle, any organism can serve as a host to express proteins from a different species:

A

bacteria
yeast
insects and insect viruses
mammalian cells in culture

46
Q

the most common hosts for protein expression

A

bacteria

47
Q

advantages of using bacterial hosts

A

-regulatory sequences are well understood
-can express high levels of cloned proteins
-easy to store and grow
-efficient methods for transforming and extracting DNA
-can be grown in huge amounts

48
Q

disadvantages of using bacterial hosts

A

-some heterologous proteins do not fold correctly
-proteins may not undergo necessary posttranslational modifications or proteolytic cleavage
-some gene sequences can be difficult to express
–many eukaryotic proteins aggregate into insoluble cellular precipitates (INCLUSION BODIES)

49
Q

transcription from the lac promoter

A

-gene of interest fused to lactose operon promoter and regulatory sequences
-transcription occurs when lactose is added to the medium
-regulation is “leaky”

50
Q

transcription from the bacteriophage T7 promoter and RNA polymerase

A

-cloned gene is fused to a T7 promoter and transcribed by T7 RNA polymerase
-affords tight regulation

51
Q

principles underlying protein expression in yeast are the same as those for bacteria:

A

-cloned genes must be linked to appropriate promoters
-gene expression can be controlled by choosing an appropriate medium

52
Q

advantages of using yeast hosts

A

-well-understood eukaryotic organism
-expression of eukaryotic genes can be more efficient
-proteins may be folded and modified more accurately

53
Q

disadvantages of using yeast hosts

A

-heterologous proteins may not fold properly
-yeast may lack the enzymes needed to modify the proteins to their active forms
-certain features of the gene sequence may hinder expression of a protein

54
Q

baculoviruses

A

insect viruses with double-stranded DNA genomes
-virus cannot make viral protein because no capsid

55
Q

bacmids

A

large circular DNAs that include the entire baculovirus gene and sequences that allow replication of the bacmid in E.coli
-store genetic material for virus without making the virus

56
Q

transfection

A

term used when the DNA used for transformation includes viral sequences and leads to viral replication
(recombinant bacmids are transfected into insect cells)

57
Q

advantages of using bacmid systems

A

-wide range available commercially
-may successfully replicate protein-modification patterns and produce active, correctly modified proteins

58
Q

disadvantages of using bacmid systems

A

not successful with all proteins

59
Q

mammalian cells in culture

A

DNA introduced into mammalian cells using engineered mammalian viruses as vectors

60
Q

advantages of mammalian cells in culture

A

-proteins can be expressed either transiently or permanently
-proper posttranslational modification can be ensured

61
Q

disadvantages of mammalian cells in culture

A

super expensive

62
Q

site-directed mutagenesis

A

technique used to individually replace specific amino acids
-alters the protein
-pcr and cleavage based

63
Q

oligonucleotide-directed mutagenesis

A

technique used to create a specific DNA sequence change
-amplify ENTIRE plasmid, then degrade old DNA

64
Q

deletions

A

performed by cutting out a segment with restriction endonucleases and ligating the remaining portion

65
Q

fusion protein

A

product of a ligated gene containing parts of two different genes

66
Q

tag

A

peptide or protein that binds a simple, stable ligand with high affinity and specificity
-fused to gene encoding target protein
-permits purification by affinity chromatography

67
Q

use of tagged proteins in protein purification

A

-provides good yield and high purity
-may affect the properties of attached proteins

68
Q

RT-PCR

A

reverse transcriptase PCR
uses reverse transcriptase to generate a DNA strand from an RNA template, followed by standard PCR protocols using DNA polymerase

69
Q

qPCR

A

quantitative PCR or real time PCR is used to estimate relative copy numbers of particular sequences in a sample
AMOUNTS

70
Q

DNA library

A

collection of DNA clones
specialized catalogs of genetic information

for
1. gene discovery
2. determination of gene or protein function

71
Q

complementary DNAs (cDNAs)

A

double-stranded DNA fragments formed from mRNA templates
-relies on reverse transcriptase
-assume its from mRNA

72
Q

cDNA library

A

population of clones created by inserting cDNA fragments into vectors and cloning

73
Q

combinatorial gene libraries

A

library focusing on sequence variants within one gene

74
Q

three levels of protein function

A

phenotypic function
cellular function
molecular function

75
Q

phenotypic function

A

describes the effects of a protein on the entire organism

76
Q

cellular function

A

describes the network of interactions a protein engages in at the cellular level

77
Q

molecular function

A

describes the precise biochemical activity of a protein

78
Q

transcriptome

A

the entire complement of transcribed RNAs present at a given moment in the cell

79
Q

proteome

A

the entire complement of proteins present at a given moment in the cell

80
Q

comparative genomics

A

process by which GENE FUNCTIONS can be assigned by using genome databases to perform genome comparisons

BLAST

81
Q

genome annotation

A

converts the sequence of residues into useful information about the location and function of genes and other critical sequences

82
Q

orthologs

A

genes that occur in different species but have a clear sequence and functional relationship to each other

83
Q

paralogs

A

genes similarly related to each other within a single species

84
Q

syteny

A

conserved gene order
-provides additional evidence for an orthologous relationship between genes at identical locations within the related segments

ex: human and mouse

85
Q

RNA -seq

A

method that determines the RNAs that are transcribed from a genome under a given set of conditions

86
Q

mass spectrometry

A

can accurately catalog and quantify the thousands of PROTEINS present in a typical cell
-complementary approach to RNA-seq
-provides information about how proteins are modified

87
Q

green fluorescent protein (GFP)

A

jellyfish protein that serves as a useful location marker
-a target gene fused to the GFP gene generates a highly fluorescent fusion protein
-variants of other colors and characteristics also exist

88
Q

IF

A

alternative approach for visualizing the endogenous protein that involves fixation (and death) of the cell

where the protein is

89
Q

epitope tag

A

short protein sequence that is bound tightly by an antibody

90
Q

knowing what a protein interacts with can suggest its function

A

the association of a protein of unknown function with one whose function is known can imply a functional relationship

techniques:
-purification of protein complexes (IP)
-yeast two-hybrid analysis

91
Q

IP

A

process of precipitating a fusion protein (containing the gene of interest and a gene for an epitope tag) by antibodies to the epitope
-proteins that bind to the tagged protein will also precipitate

92
Q

yeast two hybrid analysis

A

technique that relies on the properties of the Gal4 protein
-the two domains of Gal4pm must be brought together to function corectly
-probes molecular interactions in vivo

if you do not know binding partner *

93
Q

CRISPR/CAS systems

A

how we make mutations now

mutating or deleting a gene provides a path to understanding a gene’s function

94
Q

CRISPR/ Cas systems definition

A

specific for eukaryotic and mammalian- bacteria has a lot of other options so this is less common
“clustered, regularly interspaced short palindromic repeats”

95
Q

CRISPR sequences

A

regularly spaced short repeats in the bacterial genome, surrounding sequences derived from phage pathogens that previously infected the bacterium
cas protein= nuclease

96
Q

crispr sequences and cas protein are components of a

A

bacterial immune system

97
Q

components of the crispr/cas complex:

A

guide RNAs= transcribed viral spaced sequences that are cleaved
trans-activating CRISPR RNA (tracrRNA)
1+ Cas proteins

the complex binds and destroys invading bacteriophage DNA by the Cas protein nuclease activities

98
Q

transposons

A

segments of DNA that can move from one location to another in the genome

Biochemistry (8 decks)

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