Comparative and Functional Genomics

Question 1

What does the genome encompass?

Answer 1

All DNA in a cell, including functional genes, acquired genes, and pseudogenes

Question 2

What is the transcriptome?

Answer 2

all RNAs in a cell

Question 3

Why is the transcriptome considered dynamic?

Answer 3

because RNA expression changes frequently and doesn’t always affect phenotype

Question 4

What is the proteome?

Answer 4

the complete set of proteins produced by a cell

Question 5

Which ‘ome’ most directly determines phenotype?

Answer 5

proteome

Question 6

What technique is based on hybridisation of DNA?

Answer 6

Southern blot

Question 7

What is applied to make DNA single stranded in Southern Blotting?

Answer 7

NaOH

Question 8

What labels are used on probes in southern blots?

Answer 8

radioactive labels

Question 9

What is a major limitation of southern blotting?

Answer 9

it only detects gene presence, not extra genes or mutations

Question 10

What causes cross-hybridisation in southern blots?

Answer 10

similar sequences binding weakly

Question 11

What applies oligonucleotide dots onto microarrays?

Answer 11

inkjet primers

Question 12

What reduces cross-hybridisation in microarrays?

Answer 12

use of small, unique DNA fragments

Question 13

What enzyme extends hybridised primers in genomic microarrays?

Answer 13

Klenow DNA polymerase

Question 14

What do green, red, and yellow dots represent in two-genome microarrays?

Answer 14

green = genome A, red = genome B, yellow = both genomes

Question 15

What does orange indicate on a microarray dot?

Answer 15

both genomes hybridised, but genome B more strongly

Question 16

What is one major drawback of comparative genomics alone?

Answer 16

it cannot confirm gene function

Question 17

What does Northern blotting measure?

Answer 17

mRNA abundance

Question 18

What is used as a control in Northern blotting?

Answer 18

a housekeeping gene

Question 19

Can Northern blots be re-probed like Southern blots?

Answer 19

yes

Question 20

What is a key limitation of Northern blotting?

Answer 20

it analyses only one gene at a time

Question 21

What converts mRNA into cDNA in RT-PCR?

Answer 21

reverse trasncriptase

Question 22

Why is RNAse H activity useful in RT-PCR?

Answer 22

it degrades RNA to prevent re-synthesis of the same cDNA

Question 23

What must be avoided for accurate quantification in PCR?

Answer 23

exiting the linear phase of amplification

Question 24

How are results visualised in RT-PCR?

Answer 24

by band intensity on a membrane

Question 25

What are mRNAs converted to in transcriptomic microarrays?

Answer 25

stable cDNAs

Question 26

What labels are used in transcriptomic microarray primers?

Answer 26

Fluorescent dyes

Question 27

What kind of primers are used to capture all mRNAs?

Answer 27

random hexamers

Question 28

What is the required fold-change for a positive microarray result?

Answer 28

2-fold difference

Question 29

What are transcriptomic microarrays used to compare?

Answer 29

gene expression between two conditions

Question 30

Is transcriptomic microarray data fully quantitative?

Answer 30

No, it's semi-quantitative

Question 31

Why are many controls needed in transcriptomics?

Answer 31

due to multiple points of potential failure

Question 32

Are transcriptomic experiments expensive or cheap?

Answer 32

expensive

Question 33

do mRNA changes always reflect phenotype?

Answer 33

no

Question 34

Can mRNA intensity be compared between genes?

Answer 34

no, only between conditions for the same gene

Question 35

What two properties are proteins separated by in 2D gels?

Answer 35

isoelectric point and molecular weight

Question 36

What is the first separation step in 2D gels?

Answer 36

isoelectric focusing

Question 37

What is applied to coat proteins with negative charge in 2D gel electrophoresis?

Answer 37

SDS

Question 38

What is used to visualise proteins in 2D gel electrophoresis?

Answer 38

silver staining

Question 39

What's a challenge in analysing 2D gel images?

Answer 39

overlaying and aligning different samples

Question 40

How many proteins can typically be resolved in 2D gels?

Answer 40

around 300

Question 41

What types of proteins are typically lost in 2D gels?

Answer 41

membrane proteins

Question 42

What pH extremes are problematic in 2D gels?

Answer 42

below 3 and above 10

Question 43

What improvement does 2D-DIGE offer?

Answer 43

samples are labelled and run on the same gel

Question 44

Does 2D-DIGE fix membrane protein loss?

Answer 44

no

Question 45

What enzyme digests proteins before mass spectrometry?

Answer 45

protease

Question 46

What is generated from mass spectrometry of proteins?

Answer 46

a mass fingerprint

Question 47

What database helps identify proteins from MS?

Answer 47

A theoretical fingerprint database

Question 48

What is the advantage of robotic spot picking in mass spectrometry?

Answer 48

reduces contamination

Question 49

What separates peptides in LC-MS/MS before MS analysis?

Answer 49

liquid chromatography

Question 50

What is measured to determine abundance in LC-MS/MS?

Answer 50

absorbance at 212nm

Question 51

How many mass spectrometry steps are in LC-MS/MS?

Answer 51

two

Question 52

What is a key advantage of LC-MS/MS over 2D gels?

Answer 52

better for membrane proteins

Question 53

How many proteins can LC-MS/MS identify?

Answer 53

up to 3,000

Question 54

What type of computer analysis is used in LC-MS/MS?

Answer 54

abundance tables

Question 55

Which 'ome' is the most dynamic?

Answer 55

transcriptome

Question 56

What method provides presence/ absence of genes but not function?

Answer 56

comparative genomics

Question 57

Which technique uses fluorescence to quantify gene expression?

Answer 57

microarray transcriptomics

Question 58

Which proteomic technique allows two samples to be directly compared on one gel?

Answer 58

2D-DIGE

Question 59

Why can't comparative genomics alone confirm a hypothesis?

Answer 59

it doesn't show gene expression or protein function