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BIO 315 > Module 7 - Microbial Genomics > Flashcards

Module 7 - Microbial Genomics Flashcards

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1
Q

What is genomics?

A

Methods to study the entire genome of a microbe

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2
Q

Why is genomics important?

A

It provides insight into evolutionary relationships and detection of unknown organisms

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3
Q

What does introduction of DNA into microbes often lead to?

A

Gene disruption, allowing for studying loss-of-function on phenotype

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4
Q

What is a genome?

A

An organism’s complete set of DNA (including all genes)

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5
Q

What is genomics?

A

The collective characterization and quantification of genes (entire genome)

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6
Q

What is genomics?

A

The collective characterization and quantification of genes (entire genome)

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7
Q

What does genomics focus on?

A

The structure, function, evolutionary mapping, and editing of genomes

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8
Q

What needs (3) were created based on genomics?

A
  1. Improved DNA sequencing techniques
  2. Formats for storage of very large data sets
  3. Tools for analysis of large data sets
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9
Q

What needs (3) were created based on genomics?

A
  1. Improved DNA sequencing techniques
  2. Formats for storage of very large data sets
  3. Tools for analysis of large data sets
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10
Q

When was DNA sequencing first developed?

A

1970

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11
Q

What is sequencing?

A

The process of determining nucleic acid sequence

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12
Q

What did Walter Gilbert do?

A

Developed a chemical degradation method of DNA sequencing

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13
Q

When was Sanger sequencing developed?

A

Around 1970 (same time as Gilbert sequencing)

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14
Q

When was Sanger sequencing developed?

A

Around 1970 (same time as Gilbert sequencing)

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15
Q

What did Fredrick Sanger do?

A

Developed an enzymatic method using DNA polymerase

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16
Q

What is another name for Sanger sequencing?

A

Dideoxy sequencing, or chain termination sequencing

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17
Q

What are the three steps of Sanger sequencing?

A
  1. Clone a gene of fragment of DNA of interest
  2. Synthesize DNA with DNA polymerase
  3. Use electrophoresis to separate fragments of DNA at different lengths
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18
Q

What are the three steps of Sanger sequencing?

A
  1. Clone a gene of fragment of DNA of interest
  2. Synthesize DNA with DNA polymerase
  3. Use electrophoresis to separate fragments of DNA at different lengths
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19
Q

What is Sanger sequencing based on?

A

The fact that DNA polymerase requires a free 3’ OH group to continue DNA synthesis

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20
Q

What holds the two strands of DNA together?

A

Hydrogen bonds

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21
Q

What holds two consecutive nucleotides together?

A

Phosphodiester bonds (between 5’ phosphate group and 3’ hydroxyl group)

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22
Q

What is the significance of the 3’ OH group?

A

It is essential for chain elongation

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23
Q

What is the significance of the 3’ OH group?

A

It is essential for chain elongation

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24
Q

What is a nucleotide with a 3’ H called?

A

A dideoxyribonucleoside triphosphate (ddNTP)

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25
Q

What is the principle of dideoxy sequencing?

A

ddNTPs will stop chain elongation

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26
Q

What is the principle of dideoxy sequencing?

A

ddNTPs will stop chain elongation

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27
Q

How is the DNA sequence determined in Sanger sequencing?

A

By detecting labeled nucleotide at the end of each fragment separated by gel electrophoresis

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28
Q

How is the DNA sequence determined in Sanger sequencing?

A

By detecting labeled nucleotide at the end of each fragment separated by gel electrophoresis

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29
Q

What are the components (4) of the reaction mixture for Sanger sequencing?

A
  1. A template DNA to be sequenced
  2. A short oligonucleotide primer
  3. DNA polymerase enzyme
  4. All 4 dNTPs
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30
Q

How is the primer designed in Sanger sequencing?

A

To be complementary to the vector sequence

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31
Q

Why is the primer labeled in Sanger sequencing?

A

To allow for ease of detection of DNA pieces

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32
Q

Why is the primer labeled in Sanger sequencing?

A

To allow for ease of detection of DNA pieces

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33
Q

How is the Sanger sequencing reaction carried out?

A

In 4 tubes

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34
Q

What is the different with each tube in Sanger sequencing?

A

The specific ddNTP used (ddATP, ddTTP, ddCTP, and ddGTP)

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35
Q

What happens when a ddNTP is incorporated in a reaction in Sanger sequencing?

A

DNA synthesis is terminated

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36
Q

What happens when a ddNTP is incorporated in a reaction?

A

DNA synthesis is terminated

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37
Q

What happens when a dNTP is incorporated in a reaction in Sanger sequencing?

A

The DNA chain elongation will continue

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38
Q

What determines how many products will be in each reaction tube in Sanger sequencing?

A

Based on the number of times a specific nucleotide appears in the template DNA sequence (number of incorporation points)

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39
Q

How does a gel separate DNA fragments?

A

Larger fragments are at the top, while smaller fragments are at the bottom

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40
Q

How does a gel separate DNA fragments?

A

Larger fragments are at the top, while smaller fragments are at the bottom

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41
Q

What types of labels can be used in Sanger sequencing?

A

Radioactive labels, or fluorescent labels

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42
Q

How come fluorescent labels are used more frequently than radioactive labels?

A

Fluorescent labels are safer, cheaper, and easier than radioactive labels

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43
Q

How come fluorescent labels are used more frequently than radioactive labels?

A

Fluorescent labels are safer, cheaper, and easier than radioactive labels

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44
Q

How many bases can be sequenced by Sanger sequencing in a day?

A

700-1000 bases

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45
Q

How many bases can be sequenced by Sanger sequencing in a day?

A

700-1000 bases

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46
Q

How is a gel read in Sanger sequencing?

A

From bottom to top (Guitar Hero)

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47
Q

If a gel from Sanger sequencing reads 5’-AGTCT-3’, what is the DNA sequence from 5’ to 3’?

A

5’-AGACT-3’

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48
Q

If a gel from Sanger sequencing reads 5’-AGTCT-3’, what is the DNA sequence?

A

5’-AGACT-3’

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49
Q

What is primer walking?

A

Designing primers such that the 5’ end complements the end of the last DNA segment sequenced

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50
Q

What is primer walking?

A

Designing primers such that the 5’ end complements the end of the last DNA segment sequenced

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51
Q

What is pyrophosphate?

A

Two phosphates bonded together

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52
Q

What is pyrophosphate?

A

Two phosphates bonded together

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53
Q

How is pyrosequencing similar to Sanger sequencing?

A

It uses DNA polymerase

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54
Q

What happens if a dNTP is incorporated in pyrosequencing?

A

A pyrophosphate is released, which can be detected

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55
Q

What happens if a dNTP is incorporated in pyrosequencing?

A

A pyrophosphate is released, which can be detected

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56
Q

What reaction does pyrophosphate undergo in pyrosequencing?

A

APS + PP –> ATP

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57
Q

What does APS stand for?

A

Adenosine phosphosulfate

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58
Q

What enzyme catalyzes the reaction of pyrophosphate in pyrosequencing?

A

ATP-sulfurylase

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59
Q

What does ATP-sulfurylase do?

A

Converts APS and pyrophosphate into ATP

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60
Q

What does ATP do in pyrosequencing?

A

It can be used in a luciferase reaction to produce detectable light

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61
Q

What does CCD stand for?

A

Charged-coupled device

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62
Q

What does CCD stand for?

A

Charged-coupled device

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63
Q

What happens in pyrosequencing after a wash?

A

The reaction is repeated with a different dNTP base

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64
Q

When is light detected in pyrosequencing?

A

Only when the dNTP is complementary to the template base

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65
Q

How many bases can be sequenced by pyrosequencing in a day?

A

300-500

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66
Q

What is pyrosequencing used for?

A

Resequencing or sequencing genomes for which a close relative is already available

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67
Q

What is pyrosequencing used for?

A

Resequencing or sequencing genomes for which a close relative is already available

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68
Q

What are the steps of whole-genome shotgun sequencing?

A
  1. Shear the DNA into short pieces
  2. Sequence the fragments
  3. Use a computer algorithm to reconstruct
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69
Q

What are the steps of whole-genome shotgun sequencing?

A
  1. Shear the DNA into short pieces
  2. Sequence the fragments (Sanger sequencing)
  3. Use a computer algorithm to reconstruct
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70
Q

How can the fragments of DNA be sequenced in whole-genome shotgun sequencing?

A
  1. Cloning fragments, then Sanger sequencing

2. Directly on fragments using high-throughput sequencing

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71
Q

What do computer programs do in whole-genome shotgun sequencing?

A

Identify regions of sequence overlap from the fragments

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72
Q

What do computer programs do in whole-genome shotgun sequencing?

A

Identify regions of sequence overlap from the fragments

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73
Q

How come ~10x genome size is needed for whole-genome shotgun sequencing?

A

There is a random distribution of fragments generated

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74
Q

How come ~10x genome size is needed for whole-genome shotgun sequencing?

A

There is a random distribution of fragments generated

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75
Q

How many microorganisms have currently been sequenced?

A

3,000

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76
Q

How many microorganisms have currently been sequenced?

A

3,000

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77
Q

What is another name for high-throughput sequencing?

A

Next-generation sequencing

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78
Q

How many base pairs can high-throughput sequencing sequence at a time?

A

25-500

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79
Q

How many reads can high-throughput sequencing generated?

A

Hundreds, thousands, or millions of reads

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80
Q

What is the consequence of high-throughput sequencing having many reads?

A

High coverage, but a more computationally intensive assembly process

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81
Q

What are some examples of high-throughput sequencing?

A

Illumina (Solexa), Nanopore DNA sequencing, single molecule real time (SMRT), DNA nanoball, and SOLiD sequencing

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82
Q

What are some examples of high-throughput sequencing?

A

Illumina (Solexa), Nanopore DNA sequencing, single molecule real time (SMRT), DNA nanoball, and SOLiD sequencing

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83
Q

What DNA sequencing is currently under active development?

A

Third-generation sequencing

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84
Q

How does third-generation sequencing work?

A

By reading nucleotide sequences at the single molecule level

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85
Q

What is bioinformatics?

A

An interdisciplinary field that develops software and methods to understand large and complex biological data

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86
Q

What is bioinformatics?

A

An interdisciplinary field that develops software and methods to understand large and complex biological data

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87
Q

What is the purpose of annotation?

A

It allows for researchers to predict ORFs

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88
Q

What does ORF stand for?

A

Open reading frames

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89
Q

What is the importance of ORFs?

A

They allow researchers to better determine the start and stop points for a given gene

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90
Q

How can functions for newly discovered proteins be suggested?

A

Based on observed similarities

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91
Q

What can researchers speculate about a gene by looking at its sequence?

A

Whether it is a transcriptional factor, transport protein, or some enzyme

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92
Q

True or false: many genes have known proteins associated with them

A

False: many genes predicted by sequencing data encode gene products whose functions remain unknown

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93
Q

What is functional genomics?

A

The study of finding out the biological role of unknown genes

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94
Q

What is functional genomics?

A

The study of finding out the biological role of unknown genes

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95
Q

What types of experiments are part of functional genomics?

A

Using metagenesis to study phenotypes

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96
Q

How is gene expression generally regulated?

A

At the transcriptional level

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97
Q

What is a genomic library?

A

A collection of all the genes in a genome (cloned DNA fragments)

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98
Q

What can a genomic library be used for?

A

Whole genome sequencing

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99
Q

What does the method for obtaining a genomic library depend on?

A

The desired outcome

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100
Q

How can an mRNA library be formed?

A

By using cDNA generated from reverse transcriptase

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101
Q

What does reverse transcriptase do?

A

Convert RNA into DNA

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102
Q

What does cDNA stand for?

A

Complementary DNA

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103
Q

What does cDNA do?

A

It is the complement to the expressed mRNA

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104
Q

How can a true genomic library be formed?

A

By shearing the genome and cloning the fragments

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105
Q

How are DNA fragments prepared for a genomic library?

A

Through restriction analysis

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106
Q

How are clones generated for a genomic library?

A

By ligating the DNA fragments into plasmids, and transforming the cells

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107
Q

What determines how many clones are needed to represent the whole genome in a genomic library?

A

The size of the genome, and the average size of each cloned fragment

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108
Q

What equation can be used to calculate how many clones are needed for a genomic library?

A

N = ln(1-p)/ln(1-f)

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109
Q

In the equation for a genomic library, what is N?

A

The number of cloned fragments

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110
Q

In the equation for a genomic library, what is p?

A

The probability of generating a complete library

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111
Q

In the equation for a genomic library, what is f?

A

The average size of a fragment divided by the total genome size

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112
Q

What is the difference between a cDNA library and a true genomic library?

A

A true genomic library has all the genes, while the cDNA library only has genes that encode proteins

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113
Q

What is the difference between a cDNA library and a true genomic library?

A

A true genomic library has all the genes, while the cDNA library only has genes that encode proteins

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114
Q

What understanding do expression patterns allow for?

A

How organisms function under different conditions

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115
Q

What is a transcriptome?

A

A set of transcribed mRNA molecules in a cell

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116
Q

How can a transcriptome be measured?

A

Through northern blots

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117
Q

What is the procedure for a northern blot?

A
  1. Total RNA is isolated from cells of interest, and separated with gel electrophoresis
  2. RNA is transferred to a membrane
  3. RNA is probed with labeled DNA fragments
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118
Q

What is the procedure for a northern blot?

A
  1. Total RNA is isolated from cells of interest, and separated with gel electrophoresis
  2. RNA is transferred to a membrane
  3. RNA is probed with labeled DNA fragments
119
Q

What happens after gel electrophoresis in a northern blot?

A

RNA is transferred to a membrane made of nitrocellulose or nylon

120
Q

How is RNA linked to the nitrocellulose membrane?

A

Through exposure to UV light

121
Q

What probe is used in a northern blot?

A

A labelled DNA probe which is complementary to the mRNA being examined

122
Q

How can the location of the signal on the membrane be measured in a northern blot?

A

Through autoradiography or photography

123
Q

What is the disadvantage of a northern blot?

A

It is cumbersome to track the expression of multiple genes at once

124
Q

What is the disadvantage of a northern blot?

A

It is cumbersome to track the expression of multiple genes at once

125
Q

What is a DNA microarray?

A

A collection of microscopic DNA spots attached to a solid surface

126
Q

How does the procedure of a microarray compare to northern blotting?

A

A microarray is essentially the reverse procedure

127
Q

How does the procedure of a microarray compare to northern blotting?

A

A microarray is essentially the reverse procedure

128
Q

What are the steps of the microarray procedure?

A
  1. A glass slide is spotted with synthesized oligonucleotides
  2. Total mRNA is extracted from the cell
  3. mRNA is converted into cDNA, incorporating a fluorescent label
  4. The cDNA is passed over the chip
129
Q

In the microarray procedure, what happens if more mRNA copies are present in a sample?

A

More fluorescently labeled cDNA corresponding to that mRNA is produced

130
Q

How are the fluorescent labels used in a microarray?

A

They are captured by a scanner and analyzed by a software to quantify the amount of binding to each individual spot

131
Q

How are the fluorescent labels used in a microarray?

A

They are captured by a scanner and analyzed by a software to quantify the amount of binding to each individual spot

132
Q

How can a microarray be used with two different samples?

A

The cDNA from two different samples can be labeled differently, and hybridize to a single microarray

133
Q

How is the relative amount of cDNA in a microarray seen?

A

Through color output

134
Q

How is the relative amount of cDNA in a microarray seen?

A

Through color output

135
Q

What does Yersinia pestis do?

A

Infects a variety of rodents, and can replicate within fleas

136
Q

What is the primary transmission of Yersinia pestis between rodents?

A

Flea bites

137
Q

How can Yersinia pestis be transmitted to humans?

A

Via fleas or an infected animal

138
Q

What is special about the hosts for Yersinia pestis?

A

They differ greatly in internal body temperature

139
Q

What is the internal body temperature of a human

A

37 C

140
Q

What is the internal body temperature of a flea?

A

26 C

141
Q

What conditions were used in the microarrays studying Yersinia pestis?

A

Two different temperatures, corresponding to the flea and human

142
Q

What can be done to the genes identified from the Yersinia pestis microarray?

A

They can be studied further to identify function, role in pathogenesis, or potential as targets for therapeutic applications

143
Q

What can be done to the genes identified from the Yersinia pestis microarray?

A

They can be studied further to identify function, role in pathogenesis, or potential as targets for therapeutic applications

144
Q

What is the most recent technology in studying transcriptomes?

A

RNA-sequencing

145
Q

What is another name for RNA-sequencing?

A

RNA-seq

146
Q

What are the steps for RNA-seq?

A
  1. RNA is isolated from cells, and the rRNA is removed
  2. Remaining RNA is converted to cDNA by reverse transcriptase
  3. The cDNA can be sequenced through high throughput sequencing
147
Q

In RNA-seq, before cDNA is sequenced, what is done?

A

Sequencing linkers are attached to cDNA fragments

148
Q

What does the cDNA represent in RNA-seq?

A

The transcripts within a cell

149
Q

What happens after the cDNA is sequenced in RNA-seq?

A

The resulting sequences are compared to known RNA sequences (bioinformatics)

150
Q

How can the abundance of RNA transcripts be confirmed in RNA-seq?

A

Through qPCR

151
Q

What was RNA-seq first used for?

A

Used to examine the transcriptome of Saccharomyces cerevisiae

152
Q

Besides microorganisms, what can RNA-seq be used on?

A

The genome of RNA viruses

153
Q

Which is used more frequently: RNA-seq or microarrays?

A

RNA-seq

154
Q

What will cause RNA-seq to evolve?

A

Advancements in DNA sequencing and analysis methods

155
Q

What is a proteome?

A

A collection of all the proteins present in a cell under specific conditions

156
Q

What do differences in protein types and abundance reflect?

A

Changes in gene expression and/or protein stability

157
Q

Why is the study of proteomes important?

A

Proteins can vary due to stability or post-translational modifications, which cannot be detected by analyzing gene expression or mRNA

158
Q

What are some methods to study proteomics?

A

2D-PAGE, mass spectrometry, X-ray crystallography, and NMR

159
Q

What does NMR stand for?

A

Nuclear magnetic resonance

160
Q

What does 2D-PAGE stand for?

A

2D-polyacrylamide gel electrophoresis

161
Q

What is 2D-PAGE?

A

A 2D separation method to separate proteins based on two properties

162
Q

What properties are proteins separated by in a 2D-PAGE?

A

Isoelectric point and mass

163
Q

What does pI stand for?

A

Isoelectric point

164
Q

What is an isoelectric point?

A

The pH where a protein has no net charge

165
Q

What determines the isoelectric point of a protein?

A

The amino acid sequence

166
Q

What will a protein do in a pH gradient?

A

Migrate to the pH that matches pI

167
Q

What do proteins in a 2D-PAGE look like?

A

Spots on a 2D matrix

168
Q

What does the pattern of spots represent on a 2D-PAGE?

A

The protein composition of a cell

169
Q

What does the different pattern of spots in a 2D-PAGE suggest?

A

A measure of gene expression in different conditions

170
Q

How are many protein in a cell expressed?

A

Under conditions where they function or are needed by the cell

171
Q

How can the identities of proteins in an individual spot in a 2D-PAGE be determined?

A

By using mass spectrometry

172
Q

What makes mass spectrometry possible for identifying proteins?

A

Being able to compare amino acid sequences with corresponding genome sequence databases

173
Q

What are the steps to identify a protein from a 2D-PAGE?

A
  1. Spots are extracted from the gel
  2. The protein is digested into smaller fragments
  3. The fragments are analyzed by mass spectrometry to determine amino acid sequence
  4. These sequences are compared to a sequence database
174
Q

How is a protein digested into smaller fragments?

A

Through proteases

175
Q

What do proteases do?

A

Break down proteins into smaller fragments

176
Q

How is the amino acid sequence determined from mass spectrometry?

A

By considering the individual mass/charge ratio

177
Q

What is comparative genomics?

A

The study of evolutionary processes using the tools of genomics

178
Q

What is the goal of comparative genomics?

A

To determine relationships between species

179
Q

What can comparative genomics identify (in terms of genes)?

A

Genes associated with virulence and pathogenicity

180
Q

Where does genetic variability come from?

A

Mutations in the DNA sequence

181
Q

What are homologs?

A

Genes in a given genome that belong to related gene families that share a common ancestral DNA sequence

182
Q

What are the two types of homologs?

A

Paralogs and orthologs

183
Q

How can homologs arise?

A

From a gene duplication event

184
Q

What happens (in terms of evolution) what a gene is duplicated?

A

One copy of the gene can evolve novel functions, while the other copy performs the original function

185
Q

What are paralogs?

A

Homologous genes that arise from a duplication event

186
Q

What is an example of a paralog?

A

Malate dehydrogenase and lactate dehydrogenase

187
Q

What is an example of a family of paralogs?

A

ABC transporters

188
Q

True or false: paralog families are tiny

A

False: they can be quite large

189
Q

What is important in the evolution of genomes?

A

The duplications of genes

190
Q

What are orthologs?

A

Homologs that evolved from the same ancestor with the same function in different organisms

191
Q

What is an example of an ortholog?

A

Malate dehydrogenase in two different genomes

192
Q

What does HGT stand for?

A

Horizontal gene transfer

193
Q

What is horizontal gene transfer?

A

The movement of DNA between organisms other than transmission from parent to offspring

194
Q

What is another name for HGT?

A

Lateral gene transfer

195
Q

True or false: the genome of every organism contains foreign genes

A

True: HGT is important in all organisms

196
Q

True or false: plasmids are the only way HGT can occur

A

False: the genome of every organism contains foreign genes

197
Q

What is an indication that a gene is foreign?

A

If the base pair composition differs significantly from the rest of the chromosome

198
Q

What is a characteristic of a specific genome (specific to a particular organism)?

A

The GC content

199
Q

What is the GC content of E. coli?

A

50%

200
Q

What is the GC content of Streptomyces coelicolor?

A

72%

201
Q

What is the GC content of S. cerevisiae?

A

38%

202
Q

What is some evidence for HGT?

A

Gene phylogeny and differences in nucleotide pair patterns

203
Q

What was found when comparing genome sequences of related microbes?

A

Large segments of DNA exist in one genome, but not in a closely related genome

204
Q

What do large segments of DNA that exist in one genome, but not a closely related one, suggest?

A

Introduction or removal of large stretches of DNA occur through one or more gene transfer mechanisms with the assistance of transposable elements

205
Q

What are genomic islands?

A

Introduced DNA segments greater than 10kb up to 200 kb

206
Q

How are genomic islands detected?

A

By comparison of genomic sequences and analysis of nucleotide composition

207
Q

What is a characteristic of genomic islands?

A

They often contain GC content different from the majority of the genome

208
Q

What are genomic islands usually associated with?

A

tRNA genes, transposable elements, plasmids, or bacteriophages

209
Q

What is metagenomics?

A

A process where DNA is extracted directly from microbial communities and analyzed as a composite mixture

210
Q

What happens to extracted DNA in metagenomics?

A

They can be sequenced directly or cloned into vectors to make libraries for further analysis

211
Q

What is another name for metagenomics?

A

Environmental genomics

212
Q

What is the consequence of having many organisms in microbial communities?

A

It is difficult to complete DNA sequences of each individual genome

213
Q

How come only a subset of microbial communities are targeted for metagenomics?

A

There are many organisms in microbial communities

214
Q

In what environments is metagenomics used?

A

Freshwater, wastewater treatment systems, acid mine drainages, and deep-sea thermal vents

215
Q

What can metagenomics lead to?

A

The discovery of new genes encoding novel enzymes and antibiotics, or evidence of new organisms

216
Q

What are the steps for metagenomics?

A
  1. Obtain DNA from a sample
  2. Sequence using rapid next-gen sequencing
  3. Analyze sequences using a computer
217
Q

What must be done to analyze sequences in metagenomics?

A

Sequences from already known organisms must be eliminated

218
Q

In Sanger sequencing, which dNTP is radiolabeled?

A

dCTP (32P-dCTP)

219
Q

What is the importance of radiolabeling dCTP?

A

It is important for collecting data

220
Q

True or false: radiolabeled dCTP can be added to the DNA strand

A

True: it still has the 3’ OH group

221
Q

What helped increase the number of nucleotides that can be read from Sanger sequencing?

A

Improvements in gel electrophoresis technology

222
Q

What were some improvements of Sanger sequencing over the years?

A

Thermostable polymerases, fluorescent labels for each ddNTP, and base-calling softwares

223
Q

How are thermostable polymerases advantageous for Sanger sequencing?

A

It allows multiple rounds of synthesis from a single template strand

224
Q

How are fluorescent labels for each ddNTP advantageous for Sanger sequencing?

A

The reaction can be done in one tube instead of four

225
Q

How are base-calling softwares advantageous for Sanger sequencing?

A

They can interpret the raw data automatically and provide a direct sequence output

226
Q

What is automated sequencing?

A

Using longer electrophoresis in the Sanger reaction to lead to more sequencing data

227
Q

What is another name for automated sequencing?

A

Cycle-sequencing

228
Q

When was high-throughput sequencing developed?

A

2004

229
Q

What does apyrase do?

A

Removes unincorporated dNTPs and ATP from the system

230
Q

Where is apyrase used?

A

In pyrosequencing

231
Q

What is 454-pyrosequencing?

A

An adaptation of pyrosequencing that dramatically increased throughput

232
Q

Why is it called 454-pyrosequencing?

A

“454” is the code name of the sequencing technology project

233
Q

What are the steps of 454-pyrosequencing?

A
  1. DNA is fragmented into short pieces, then ligated onto beads
  2. Each bead can be separated into its own PCR reaction
  3. The pyrosequencing reagents are added to each well
234
Q

What is the advantage of 454-pyrosequencing?

A

It increases sequencing throughput

235
Q

What is emulsion PCR?

A

PCR carried out on the surface of a bead in an oil emulsion

236
Q

Where is emulsion PCR used?

A

In 454-pyrosequencing

237
Q

What is Ion Torrent?

A

A high-throughput sequencing method

238
Q

What is Ion Torrent related to?

A

454-pyrosequencing

239
Q

How does Ion Torrent work?

A

Like 454-pyrosequencing, but it measures protons instead of pyrophosphate each time a nucleotide is incorporated

240
Q

What is another name for Ion Torrent?

A

Ion-semiconductor sequencing

241
Q

How does Illumina sequencing work?

A
  1. DNA polymerase adds fluorescently labeled nucleotides
  2. An image is taken, and the fluorescent label is cleaved
  3. Paired-end reads can be analyzed separately
242
Q

What is PacBio?

A

A system for genome sequencing

243
Q

What is the advantage of more coverage in shotgun sequencing?

A

It helps reduce gaps in the final assembly that would need to be filled in with time consuming methods like primer walking

244
Q

How can gaps be reduced in shotgun sequencing?

A

By combining a long, error-prone sequencing with short sequences

245
Q

In shotgun sequencing, which method produces long, error-prone sequences?

A

PacBio

246
Q

In shotgun sequencing, which method produces short sequences?

A

Ion Torrent or Illumina

247
Q

What does the long error-prone sequence do in shotgun sequencing?

A

Gives a backbone (scaffold) for the overall genome

248
Q

What do the short sequences do in shotgun sequencing?

A

Ensure that the final genome does not contain errors

249
Q

What is a pan-genome?

A

The full complement of all genes within a species of bacteria (in all different strains)

250
Q

What fundamental technique is used to study the transcriptome?

A

Nucleic acid hybridization

251
Q

What does a Southern blot do?

A

Detects specific DNA sequences

252
Q

What are the steps for a Southern blot?

A
  1. Generate DNA fragments through restriction enzymes
  2. Separate the fragments through gel electrophoresis
  3. Transfer the DNA fragments to a membrane for further analysis
253
Q

What advances led to DNA microarrays?

A

Advances in photolithography and the availability of complete genome sequences

254
Q

What does photolithography refer to?

A

The technique of creating computer chips

255
Q

In the Y. pestis experiment, which genes could be important in pathogenicity?

A

The green and red genes (not the yellow genes)

256
Q

How come the green and red genes may be important in Y. pestis pathogenicity?

A

They respond differently according to the different host temperatures

257
Q

What does SDS-PAGE stand for?

A

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

258
Q

What does an SDS-PAGE do?

A

Separates denatured proteins based on mass

259
Q

How does an SDS-PAGE work?

A

It mitigates charge, so proteins can be separated based on mass alone

260
Q

How come most of the proteome would not resolve on an SDS-PAGE?

A

Many polypeptides have similar mass

261
Q

What are the steps of a 2D gel?

A
  1. Apply a protein sample to a pH gradient on a polyacrylamide strip
  2. Use a current to separate by pI
  3. Use this strip in electrophoresis to separate by mass
262
Q

What does LC-MS stand for?

A

Liquid chromatography-mass spectrometry

263
Q

What is the purpose of LC-MS?

A

To separate proteins by LC before analyzing them with MS

264
Q

What methods can be used to study the structures of proteins?

A

X-ray crystallography and NMR

265
Q

How does X-ray crystallography work?

A

Crystallized proteins are subjected to X-rays. The scattering patterns can be detected and analyzed

266
Q

How easy is protein crystallization?

A

It is a delicate and often difficult process

267
Q

What are the disadvantages of protein crystallization?

A

Not all proteins crystalize well

268
Q

Which proteins are especially difficult to crystallize?

A

Hydrophobic proteins that normally associate with cytoplasmic membranes

269
Q

What is the advantage of NMR?

A

It is able to determine the structure of proteins while in solution

270
Q

How does NMR work?

A

It measures the distances between atomic nuclei

271
Q

What is the disadvantage of NMR?

A

It has a maximal size limit of about 30 kDa (the size of an average bacterial protein)

272
Q

How are the different proteins in a paralog family related?

A

They probably carry out similar functions, but on different substrates

273
Q

How are the different proteins in an ortholog family realted?

A

They probably carry out the same function (and same substrate) in different organisms

274
Q

What does the evolutionary relationship of orthologs mirror?

A

The evolutionary history of their respective genomes

275
Q

What do dehydrogenases do?

A

They carry out NAD or NADP dependent reactions

276
Q

What does malate dehydrogenase do?

A

Converts malate into oxaloacetate

277
Q

What does lactate dehydrogenase do?

A

Converts lactate into pyruvate

278
Q

Why can genomes be considered as mosaics?

A

They have arisen from evolutionary changes and horizontal gene transfer

279
Q

How come scientists first believed that HGT had no evolutionary advantage to microbes?

A
  1. There were different gene regulatory elements involved
  2. Expression was needed for the new gene
  3. The new gene could be detrimental
280
Q

Besides GC content, what are some clues that HGT has taken place?

A

Differences in patterns of nucleotide base pairs, codon usage patterns, and presence of repetitive sequences

281
Q

How can gene phylogeny imply HGT?

A

If evolutionary relationships from the gene do not match the core genome, this is evidence that HGT has taken place

282
Q

When is HGT most successful?

A

When two organisms have similar DNA and GC content

283
Q

What are some examples of advancements due to metagenomics?

A
  1. The discovery of proteorhodopsins
  2. The discovery of ammonia-oxidizing archaea
  3. The discovery of vitamin B12 producers in the ocean
284
Q

What do proteorhodopsins do?

A

Harness light energy for metabolism in marine environments

285
Q

Who pioneered functional metagenomics?

A

Jo Handelsman

286
Q

How can sequences from metagenomics be linked to specific microbes?

A

Through databases or SSU rRNA genes

287
Q

What are some limitations of metagenomics?

A

Researchers cannot make firm predictions based solely on available sequence information

288
Q

What does FACS stand for?

A

Fluorescently associated cell sorting

289
Q

How does FACS work?

A

A DNA probe for a SSU rRNA is used in a permeabilized cell to recover individualized uncultivated cells

290
Q

What is single-cell genomics?

A

Understanding the genome of one cell through FACS

291
Q

What are some challenges of metagenomics?

A

Co-extracted contaminants and community complexity

292
Q

What is metatranscriptomics?

A

The study of RNA transcripts directly extracted from a commuinty

293
Q

What is metaproteomics?

A

The study of environmental proteins

294
Q

How do metatranscriptomics and metaproteomics work?

A

Through bioinformatics (similar to individual organisms)

BIO 315 (12 decks)

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