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Nucleic Acids and Proteins > Nucleic Acids and Proteins > Flashcards

Nucleic Acids and Proteins Flashcards

1
Q

Degrade DNA molecules by breaking the phosphodiester bonds that link one nucleotide to the next in a DNA strand.

A

nucleases

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2
Q

Two kinds of nucleases

A

Exonuclease
Endonuclease

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3
Q

Nucleases vary in

A

Specificity

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4
Q

May be specific for DNA or RNA, such as DNases or RNases, respectively, or even be specific for a DNA/RNA hybrid, such as RNase H, which cleaves the RNA strand of a DNA-RNA hybrid. Therefore, its specificity varies dramatically.

A

nucleases

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5
Q

Remove nucleotides one at a time from the end of a DNA molecule

A

Exonuclease

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6
Q

May either attack a polynucleotide chain from the 5’ end and hydrolyze 5’ to 3’ or attack from the 3’ end and hydrolyze 3’ to 5’

A

Exonuclease

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7
Q

When a nuclease hydrolyzes an ________________ bond in a ________________ linkage, it will have specificity for either of the two ester bonds, generating either 5’ nucleotides or 3’ nucleotides

A

ester; phosphodiester

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8
Q

Nucleases may be specific for single strand nucleotide chain, double-helix stands, or both.

A

Strand preference

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9
Q

An exonuclease that can remove nucleotides from both strands of a double-stranded molecule

A

Bal31

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10
Q

An enzyme that degrades just one strand of a double-stranded molecule, leaving single-stranded DNA as the product

A

Exonuclease III

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11
Q

Can hydrolyze internal bonds within a polynucleotide chain (break in the middle)

A

Endonuclease

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12
Q

Can attack the phosphodiester bond from the 5’ end or from the 3’ end of the linkage

A

Endonuclease

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13
Q

________________ endonuclease only cleaves single strands, whereas ________________ cuts both single- and doublestranded

A

S1; deoxyribonuclease I (DNase I)

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14
Q

Recognize a specific nucleotide sequence and cleave the DNA molecules internally

A

Restriction endonucleases

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15
Q

More commonly used in the lab

A

Restriction endonucleases

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16
Q

3 classes of restriction endonucleases are distinguished by

A

mode of action

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17
Q

Class of restriction endonuclease that are rather complex and have only a limited role in practical biotechnology applications.

A

Type I and Type III

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18
Q

Class of restriction endonucleases which are the cutting enzymes that are so important in laboratory and clinical analysis.

A

Type II

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19
Q

Type II restriction endonuclease is important because these sites (2) are the same.

A

recognition site
cleavage site

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20
Q

Length of recognition/cutting sites

A

4 to 8 bp

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21
Q

This means reading the same forward and backward on complementary strands

A

inverse palindromic

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22
Q

Different sources of Type II restriction

A

Isoschizomers
Neoschizomers
Isocaudomers

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23
Q

Q1: recognize and cut DNA at the same site

Q2: produce the same nucleotide extensions but have different recognition sites

Q3: recognize and bind to the same sequence of DNA but cleave at different positions

A

Isoschizomers;
Isocaudomers;
Neoschizomers

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24
Q

Isoschizomers species

A

BspEI from a Bacillus species

AccIII from Acinetobacter calcoaceticus

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25
Q

Neoschizomers species

A

NarI from Nocardia argentinensis

SfoI from Serratia fonticola

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26
Q

Isocaudomers species

A

NcoI from Nocardia corallina

PagI from Pseudomonas alcaligenes

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27
Q

represents a linear sequence of the sites at which particular restriction enzymes find their targets

A

Restriction map

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28
Q

Before sequencing a large stretch of DNA, this is done to locate the cutting site and examine sizes of fragments

A

Preliminary mapping

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29
Q

Q1: Restriction digest can be performed in a ________________ in the presence of all necessary components

Q2: what are these components

A

microcentrifuge tube;

template DNA, restriction enzyme, Mg2+

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30
Q

Q1: A restriction digest results in a number of ________________

Q2: the sizes of which depend on the

A

DNA fragments;

positions of the recognition sequences

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31
Q

Used to analyze restriction digested DNA fragments

A

gel electrophoresis

separated based on their size

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32
Q

A specific DNA region can be analyzed by

A

Southern blot

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33
Q

Enzymes that synthesize a new strand of DNA complementary to an existing DNA or RNA template

A

Polymerases

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34
Q

Types of polymerases

A
  1. DNA Polymerase I
  2. Klenow Fragment
  3. Taq DNA polymerase
  4. Reverse Transcriptase
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35
Q

Prepared from E. coli which an example of an enzyme with a dual activity

A

DNA polymerase I

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36
Q

DNA polymerase I activities (2)

A

DNA polymerization
DNA degradation

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37
Q

This treatment of DNA polymerase I produces two polypeptides

A

Mild proteolytic treatment

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38
Q

The large fragment produced by mild proteolytic treatment of DNA polymerase I which has the polymerase 3’ to 5’ exonuclease activity

A

Klenow fragment

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39
Q

A Klenow fragment can synthesize a complementary DNA strand on a single-stranded template also known as a ________________ region.

A

nick

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40
Q

Major application of Klenow fragment

A

Perform DNA end-filling or DNA sequencing

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41
Q

Bacterium of Taq DNA polymerase

A

Thermus aquaticus

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42
Q

Thermostable thus suitable for PCR

A

Taq DNA Polymerase

43
Q

uses RNA as a template not DNA to synthesized a complementary DNA strand

A

Reverse transcriptase

44
Q

newly synthesized DNA

A

complementary DNA (cDNA)

45
Q

Q1: Used to evaluate amount of RNA

Q2: Used to establish the expression profile (to evaluate the change of gene expression pattern)

A

Reverse transcriptase

46
Q

modify DNA molecules by addition or removal of specific chemical groups

A

DNA modifying enzymes

47
Q

DNA modifying enzymes

A

Alkaline phosphatase;
Polynucleotide kinase;
Terminal deoxynucleotidyl transferaseadds

48
Q

From calf thymus disease that adds one or more deoxyribonucleotides onto the 3’ terminus of a DNA molecule

A

Terminal deoxynucleotidly transferaseadds

49
Q

From E coli. infected with T4 phage that has reverse effect to alkaline phosphatase, adds phosphate groups onto free 5’ terminus

A

Polynucleotide kinase

50
Q

From E. coli, calf intestinal tissue, or arctic shrimp that removes the phosphate group present at the 5’ terminus of a DNA molecule

A

Alkaline phosphatase

51
Q

To simplify:

Alkaline phosphatase: removes 5’ phosphate group

Polynucleotide kinase: adds or attaches 5’ phosphate groups

Terminal deoxynucleotidyl transferaseadds: attachees deoxyribonucleotides to the 3’ termini

A
52
Q

Repair single-stranded breaks (“discontinuities”) that arise in double-stranded DNA molecules during DNA replication or during DNA damage repair

A

DNa ligase

53
Q

Joins two individual fragments of double-stranded DNA and individual DNA molecules

A

DNA ligase

54
Q

Formation of hydrogen bonds between two complementary strands of nucleic acids

A

Hybridization

55
Q

Q1: labeled strand

Q2: process called

A

Probe;
Hybridization (assay)

56
Q

Formed between a labeled and unlabed strand

A

hybrid molecule

57
Q

Reaction that is used to analyze the nucleic acid content of an unknown sample

A

hybridization assay

58
Q

Hybridization assays

A

Southern
Northern
Dot/Blot
Microarray
Fluorescent in situ

59
Q

Functions:

1) used to identify homologous sequences in genomic DNA

2) facilitate gene mapping through restriction mapping of genes

3) detection of restriction fragment length polymorphisms

A

Southern Hybridization

60
Q

Southern Hybridization process

A
  1. Extraction & Purification
  2. Enzyme Digestion (product = DNA fragments)
  3. DNA denaturation & fragmentation
  4. Transfer / Blotting
  5. Hybridization with labeled DNA probe
  6. Detection & identification
61
Q

Visualization of this means should show a series of smear bands without any discrete, distinguishable bands

A

visualized by staining

62
Q

Parameters for Southern Hybridization

A

1) resolution of the agarose gel
2) depurination of DNA molecules
3) physical transfer of DNA onto the membrane support
4) fix DNA onto the nylon membrane

63
Q

Southern Hybridization

Q1: percentage of agarose gel

Q2: voltage applied in gel electrophoresis

A

0.7 - 1.2%;

High voltage, short runs: 10-20 kbp
Low voltage, long runs:
1 and 10 kbp

64
Q

Most critical step in Southern Hybridization

A

Depurination of DNA molecules

65
Q

depurination by ________________________ upon denaturation by sodium hydroxide leads to nicks in the DNA strands, resulting in a breakdown of long DNA fragments into shorter pieces of single-stranded DNA.

A

0.2 M hydrochloric acid

66
Q

Transfer methods

A

1) Classical Southern Transfer (ascending capillary transfer)

2) Descending Capillary transfer

67
Q

Transfer of DNA to membrane in Southern Hybridization

A

Ascending capillary transfer

68
Q

Uses the gravitational flow of the transfer buffer with the help of vacuum to allow more rapid and reproducible blotting of DNA

A

Descending capillary transfer

69
Q

Fixing DNA onto the nylon membrane can be achieved by baking the membrane at or by

A

at 80C for 2 hrs

or

by UV light irradiation

70
Q

Storage for baked or UV-fixed southern blot

A

room temp

71
Q

Functions

1) allows detection of a given RNA molecules in a mixture of heterogeneous RNA

2) Uses DNA probes to hybridize with complementary RNA sequences

3) an ideal tool to study the products of gene
transcription

A

Northern Hybridization

72
Q

Northern Hybridization

Before electrophoresis, denaturation is achieved by heating sample to

A

55C in the presence of formaldehyde and formamide

73
Q

Its addition to the gel prevents reformation of secondary structure

A

formaldehyde

74
Q

not usually recommended to
add in electrophoresis, as it will always decrease
the hybridization signal compared with unstained
RNA

A

ethidium bromide

75
Q

blotting for northern hybridization

A

capillary blotting or vacuum blotting

76
Q

Rna can be fixed by

A

UV light irradiation or baking at 80C

77
Q

Denaturation time for RNa

Voltage and time

A

15 minutes

70V for 3.5 h

78
Q

Results from a variable number of tandem repeats (VNTR) in a short DNA segment

Used in DNA fingerprinting and in paternity testing

Useful as a genetic disease marker

A

Restriction Fragment Length Polymorphism (RFLP)

79
Q

performed when genomic DNA is collected and is digested with a specific restriction enzyme followed by gel electrophoresis

A

RFLP analysis

80
Q

permits the simultaneous evaluation of hundreds to thousands of different DNA regions; useful in nonmodel species

A

Amplified Fragment Length Polymorphism (AFLP)

81
Q

Allow high-resolution genotyping of fingerprinting quality

A

AFLP analysis

82
Q

Factors that influence hybrid stability

A

Ionic Strength
Base composition
Destabilizing agents
Mismatched base pairs
Duplex length

83
Q

Must be labed with a radioactive or other type of marker

A

probe

84
Q

probes are denatured by

A

heating

85
Q

applied to the membrane in a solution of chemicals that promote nucleic acid hybridization

A

Probes

86
Q

An organic molecule that has a high affinity for a protein called avidin

A

biotin

87
Q

a variation of the dot/slot blot in which the dotted material is arranged in a regular gridlike pattern

A

mircroarray

88
Q

This technology is an analysis of nucleic acid on a genome-wide scale

A

microarray

89
Q

Most comon application of microarray technology which is the gene-by-gene determination of differences

A

transcript profiling

90
Q

Process of transferring/blotting the proteins from the
gel onto the surface of an absorbent membrane to
render the proteins accessible for the identifying
ligand

A

Western Blot

91
Q

proteins are fully denatured with SDS and heat in
the presence of β-mercaptoethanol

A

SDS page

92
Q

native proteins are separated in a gel containing a pH gradient

works well for hydrophilic proteins

A

Isoelectric focusing (IFE)

93
Q

requires no buffer tank and no cooling system

A

semidry blotting

94
Q

most commonly used membrane for protein
blotting;

A

nitrocellulose

95
Q

has much superior mechanical strength than nitrocellulose;

Its protein binding capacity is good and it can be
obtained as positively charged membrane that
even increases the binding and retention capacity

A

Nylon

96
Q

Membrane of choice for many applications of Western blotting

It combines high protein binding capacity with excellent mechanical resistance and good staining properties.

A

PVDF

97
Q

Standard stains of similar sensitivity

A

Amino black and india ink

98
Q

Stain with lowest sensitivity but convenient as it is reversible by immersing in 0.1 N NaOH

A

ponceau

99
Q

gives optimal blocking with lowest background and without impairing immunoreactivity

A

Ovalbumin/gelatin

100
Q

proved to be an economical and effective blocker

A

fat free milk

101
Q

blocking is very simple but has a tendency to give elevated background; may mask or detach immunoreactive proteins

A

tween-20

102
Q

method for western blot

A

indirect immunodetection

103
Q

used in western blots which produce a colored precipitate

A

chromogen

104
Q

commonly used enzymes for western blot

A

horseradish peroxidase (HRP) and alkaline phosphatase (AP)

Nucleic Acids and Proteins (1 decks)

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