Theme 3 Flashcards
What is the transcriptome
Every RNA that can be produced from a genome
Only part of it in each cell depending on genes active
What are the 3 major classes of RNAs that make up the eukaryotic transcriptome
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
What are 3 errors that can occur during splicing
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.
What are some facts that make cDNA production work
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
What is an expressed sequence tag (EST)
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
Explain multigene analysis with DNA microarrays
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
What are 2 techniques used for transcriptomics
Multigene analysis with DNA microarrays
Deep sequencing
Explain deep sequencing
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
Explain non coding RNAs
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.
What are the steps/questions used when working out what a gene does
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?
What does evaluating gene expression entail
Finding out where and when the gene is expressed
look for transcripts i.e. RNA
Look for protein
How do we evaluate gene expression by looking for transcripts
- 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
How do we evaluate gene expression by looking for protein
- 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
What are 3 types of analysis in genomics
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?
Explain homologous genes
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
Why may identification of orthologous genes be complicated
Orthologous genes may be on different chromosomes in different species due to evolution
May be a number of similar genes (paralogues) in the genome
Explain synteny
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.
How can we use forward genetics to find gene function
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
How can we use reverse genetics to find gene function
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.
Define polymorphism, mutation and haplotype
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
Where can variations arise from
Indels
Mobile genetic elements
SNPs (90% of variation)
Outline SNP variation
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)
What is an SNP haplotype and a haplotype block
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
What does haploytyping require
A reference map of SNPs sequenced from >100 people for variation
rapid and cheap screening method to interpret SNPs
Explain an SNP array
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
Explain pharmacogenetics
Study of the relationship between genetic variation (haplotype) and response to medication
We can use haplotype variation to predict dosage and type of medication
Outline whole exome sequencing (WES)
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
