Theme 3

Question 1

What is the transcriptome

Answer 1

Every RNA that can be produced from a genome

Only part of it in each cell depending on genes active

Question 2

What are the 3 major classes of RNAs that make up the eukaryotic transcriptome

Answer 2

Ribosomal RNAs transcribed by RNA polymerase I

Protein-encoding RNAs (mRNA), and microRNAs (miRNA). Transcribed by RNA polymerase II

Small RNAs (including tRNA), transcribed by RNA polymerase III

Question 3

What are 3 errors that can occur during splicing

Answer 3

Mutations can occur or can generate new (cryptic) splice sequences

Exons can be omitted, deletes section of protein, affects protein structure

Use of false (cryptic) acceptor or donor sites can truncate or lengthen exons, affects protein structure.

Question 4

What are some facts that make cDNA production work

Answer 4

RNA can base pair with DNA

mRNA has a polyadenylated tail meaning we can always use a TTTTT primer

No pre existing gene sequence info is required

RT can convert RNA to DNA

Question 5

What is an expressed sequence tag (EST)

Answer 5

cDNAs made from mRNAs originating from a specific cell or tissue i.e. they represent a “snapshot” of mRNA present at that time and place.

Compare DNA to EST database to see if it matches a known gene in a specific cells and how much activity there is (gene verification and expression)

Can also see how many times an EST or exon is found in a specific cell (checking alternative splicing)

Unigene is used for EST matching

Question 6

Explain multigene analysis with DNA microarrays

Answer 6

Whole genome expression analysis, potentially every gene is probed

Shows where, when and how many genes expressed in specific cells

Examine patterns in normal vs disease tissues vs response to stimulus/treatment

Question 7

What are 2 techniques used for transcriptomics

Answer 7

Multigene analysis with DNA microarrays

Deep sequencing

Question 8

Explain deep sequencing

Answer 8

The produced cDNA is fragmented and amplified. Gives many short sequence reads

Compare to EST database and human genome to see where the genes came from

Question 9

Explain non coding RNAs

Answer 9

Usually we look at genes encoding proteins

There are other genes that encode non mRNA like rRNA, tRNA, snRNA, snoRNA

Involved in translation, splicing and RNA editing

microRNAs are transcription and translation regulators synthesised by RNA Pol II into a long precursor. Then processed to small mature miRNA.

Question 10

What are the steps/questions used when working out what a gene does

Answer 10

Is the end product protein or ncRNA?

Where is the gene expressed and product found?

What is the function of the product

What does the product bind to? (e.g. protein, nucleic acid, lipid)

What is its biological role?

Question 11

What does evaluating gene expression entail

Answer 11

Finding out where and when the gene is expressed

look for transcripts i.e. RNA

Look for protein

Question 12

How do we evaluate gene expression by looking for transcripts

Answer 12

• different cell expression
• changes in transcript levels or splicing

In situ hybridisation: labelled DNA or RNA complimentary to target mRNA soaked into cell/tissue. Probe binds where target mRNA is.

RT PCR: sequence of target RNA must be known for primer design. Also (q)PCR

Many transcripts at once by: Microarray, deep sequencing

Question 13

How do we evaluate gene expression by looking for protein

Answer 13

• Which cells, tissues, developmental state?
• Does level, location or activity alter in response to stimulation?

Antibodies or other binding reagent (cell / tissue structure can be maintained)

Enzyme activity (usually in cell or fluid extracts)

Mass spectrometry/proteomics (usually in cell or fluid extracts)

Can integrate reporter molecule into genome and examine by microscopy by producing a transgenic cell/animal

Question 14

What are 3 types of analysis in genomics

Answer 14

Comparative analysis: Does its sequence/structure give an idea of function?

Mutational analysis: What happens to the cell or organism if it is mutated?

Interactions: Position in pathway or network; binding partners?

Question 15

Explain homologous genes

Answer 15

Homologous genes share a common ancestor

orthologue: A gene in a separate species that has the same biological properties and function
paralogue: A related gene in the same species for which a function is known, generated by gene duplication

Question 16

Why may identification of orthologous genes be complicated

Answer 16

Orthologous genes may be on different chromosomes in different species due to evolution

May be a number of similar genes (paralogues) in the genome

Question 17

Explain synteny

Answer 17

the conservation of blocks of order within two sets of chromosomes that are being compared with each other.

Syntenic regions are where orthologous genes are likely to be located. The order of orthologous genes is conserved in syntenic blocks.

Question 18

How can we use forward genetics to find gene function

Answer 18

Phenotype to genotype

Historical/clinical: Altered trait observed, affected protein and process identified, mutated gene identified with WGS

Exploratory: Randomly mutagenise model organism, screen for interesting phenotypes and identify genes mutated. Infer how mutation causes phenotype

Question 19

How can we use reverse genetics to find gene function

Answer 19

from genotype to phenotype

Cause a loss-of-function mutation to see altered phenotype

Targeted mutation - knockout mice: Gene replaced by mutated version in ESC which are injected into normal blastocysts

Silencing gene expression by RNA interference: dsRNA (with part of target mRNA) is cleaved into short interfering siRNA by dicer enzyme. Associates with RISC and pairs with target DNA. RISC cleaves target mRNA.

CRISPR/Cas: Immune system in bacteria to cleave foreign DNA. crRNA and Cas proteins form crRNP complex. mRNA encoding Cas9 injected into cells. Cas9 cuts target DNA causing small deletions.

Question 20

Define polymorphism, mutation and haplotype

Answer 20

Polymorphism: a DNA variation present in more than 1% of people

Mutation: sequence present in less than 0.1% of people.

Haplotype: unique combination of alleles that makes up an individual

Question 21

Where can variations arise from

Answer 21

Indels
Mobile genetic elements
SNPs (90% of variation)

Question 22

Outline SNP variation

Answer 22

Theoretically 4 possible alleles but usually only 2.

In non coding region: may alter gene regulation

In coding region: could be synonymous (no effect), nonsense (prevent protein production) or missense (alter structure)

Question 23

What is an SNP haplotype and a haplotype block

Answer 23

A list of every SNP in someones genome sequence

SNPs that are linked are said to be in “linkage disequilibrium”

Haplotype block is a part of the genome usually inherited together. So could include many SNPs that are the same in offspring

Can map faster by looking at haplotype block combination instead of every SNP

Question 24

What does haploytyping require

Answer 24

A reference map of SNPs sequenced from >100 people for variation

rapid and cheap screening method to interpret SNPs

Question 25

Explain an SNP array

Answer 25

SNP microarray has a gene chip with thousands of ssDNA spanning each SNP region

Genomic DNA amplified and only binds when a perfect match occurs.

Shows if someone has same or differing SNP to probe DNA

Question 26

Explain pharmacogenetics

Answer 26

Study of the relationship between genetic variation (haplotype) and response to medication

We can use haplotype variation to predict dosage and type of medication

Question 27

Outline whole exome sequencing (WES)

Answer 27

Reference DNA shows where exons are so we just sequence the exons

Used to find difference in coding regions which can identify rare and single gene disorders