BMS2062 - W2: Genomic Variation & Diseease

Repetitive genome regions

make up ~52% of human DNA

Transposons

• capable of moving from one region of the genome to another

• can cause problems/ mutations/ alter gene expression depending on where it inserts itself into

Retrotransposons

• must first be reverse transcribed into DNA before inserting into a new genome region

Single sequence repeats

• same sequence of nucleotides repeat over and over again

• may have a structural purpose (not much is known about them)

• these regions have high mutation rate - can be indicated in disease (e.g. huntington’s disease)

Which strand is used to transcribe DNA to mRNA? Template strand or coding strand?

Template strand is used

• RNA transcript will be the same as the coding strand (except T is changed to U)

What are open reading frames?

regions between a start codon and a stop codon within a gene

How can we locate genes?

Identify presence of:

• open reading frames

• transcription/ translation control points (promoters e.g. TATA box promoter, terminators, ribosome binding sites, UTRs, enhancers, etc.)

Reading frames

there are 6 potential reading frames in DNA

What is redundancy?

Multiple codons code for the same protein. So if there is a mutation, it can still result in the same amino acid being encoded for.

Microarray vs RNA sequencing

Microarray:

• if you’re working with RNA (but not too many of them bc of limited number of probes)

• RNA → cDNA → expose cDNA to transcript probes → see which ones bind

RNA sequencing:

• can be used for many/ all RNA in a particular cell/ group of cell

• RNA → cDNA → sequencing → alignment

How can we identify the sequence of proteins?

Mass spectrometry - can be used for a single protein or a group of/ all proteins in a cell/ group of cells

Reading frame vs Open reading frame vs real open reading frames

Reading frame: Nomenclature for designating how the codons within a nucleotide sequence are read

• the set of possible codons that could code for a protein

Open reading frame: A DNA sequence that occurs between a start and a stop codon

• may or may not code for a protein

• introns need to be removed to produce the open reading frame

Real open reading frame: An open reading frame that produces a real protein (or sometimes RNA)

Bioinformatic tools

Expasy: identify reading frame

• shows all possible open reading frames

• determine reading frame

• where the open reading frame is within the reading frame

• most likely to code for a protein

protein blast: identify real reading frame

• contains all known proteins

• access if there is a match between predicted protein and proteins in the match database

How to identify open reading frame in prokaryotic DNA sequence

Bioinformatics tools discussed previously

• many stop codons = unlikely to be a prokaryotic protein coding gene region

• few stop codons + an initiation codon = likely to be a prokaryotic protein coding gene region

How to identify open reading frame in eukaryotic DNA sequence

Bioinformatic tools previously discussed

• has exon regions and intron regions - intron regions likely to have stop codons (interrupt open reading frame)

• so need to find the borders for introns and exons then find open reading frame

What is the boundary between an intron and an exon called?

Splice site

How can splice sites be detected?

There are consensus sequences for major class introns

• extract all mRNA produced by cell of a particular organism → reverse transcribe to cDNA → establish sequence of cDNA fragments → see which regions these match to in the genome → shows where exons are

• design pcr primers to bind to confirm exon/ intron boundary

What do these intron exon boundaries allow for?

multiple mRNA transcripts can be produced from a single gene (transcript variants/ isoforms) via alterative splicing

• basically different exons are incorporated to make different variants

• this increases functional capacity of genes

Homology searching

Paralogue: same species

Orthologue: different species

can gives clues about exon intron boundaries

What is the ultimate way to validate identified regions as real genes?

Find the corresponding mRNA or protein

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Transcriptomics:

reverse transcriptase - PCR

• design primers based on predicted sequences

• if the mRNA corresponding to the predicted sequence is real, it will bind to the RNA and cDNA will extend to match mRNA sequence

• detection of cDNA validates that predicted gene is real

Proteomics:

• extract the protein from cells, denature, cut them with protease

• run mas spectrometry to confirm identity of proteins

• map those back onto database

• cross check which ones are present with our predicted reading frame

]Homology modelling:

• match predicted sequence against database

Western blot

• create antibody that can recognise a specific part of protein sequence

• extract protein, denature, add antibody, add second antibody that emits light