L22-23 Flashcards
(34 cards)
2 types of genomes? Which one has less space?
Nuclear &Mitochondrial.
Mitochondrial.
Exome sequencing
Sequencing all the protein-coding genes in a genome (known as the exome). It consists of first selecting only the subset of DNA that encodes proteins (known as exons), and then sequencing that DNA using any high throughput DNA sequencing technology.
What is a transposon and where do they come from?
An active piece of DNA that can MOVE from place to place.
They come from retrotransposons (based on retrovirus sequences)
How much of the human genome is made up of transposon sequences?
40%
What do retrotransposons lack compared to retroviruses?
Lack env (envelope protein) so they cannot go outside of the cell
LINE elements (Long Interspersed Nuclear Elements)
- Occur in all mammals
- ~20% of the human genome
- Encode all components for retrotransposition (including reverse transcriptase)
- ~6kb long
- Evolutionarily related to retroviruses
SINE elements (Short Interspersed Nuclear Elements)
- ~11% of the genome (~1,500,000 copies in humans)
- Similar sequences to retroviruses but DO NOT encode their own reverse transcriptase
- Most common SINEs in primates are the Alu sequences (They contain a recognition site for the Alu restriction enzyme) - 350bp long
What are pseudogenes?
They are DNA sequences that resemble protein-coding genes but they are not transcribed to a messenger RNA (mRNA) in a way that could then be translated into some functional protein
Exon shuffling and how many % of our genes have it?
Molecular mechanism for the formation of new genes where 2 or more exons from different genes can be brought together ectopically, or the same exon can be duplicated to create a new exon-intron structure
10% of our genes have it
Domains in heavy and light chain?
Heavy chain – 4 domains
Light chain – 2 domains
Where are protein domains often found, in regards to exons?
Separate exons hence exon shuffling is more likely to produce a new protein (it’s an advantage because it makes evolution better – alternative splicing, regulatory)
How do we know which gene encodes a certain protein?
From the genome, we take possible proteins produced and put into a translated database that we compare the protein with.
What is synteny?
Regions of two genomes that is co-linear (in the same order)
What method can you use to predict the structure of a protein based on its homology?
Homology modelling
How have gene duplications allowed diversification of gene function in evolution?
Divergence of genes generates new genes
What is NCBI?
National Centre of Biotechnology Institute – people add sequences to the database
What does a BLAST search do? What different comparisons can they make?
Basic Local Alignment Search Tool which can compare different genomes.
Nucleotide blast – gene comparing to genome
Protein blast – protein comparing to protein
What does it mean to generate a sequence alignment?
Scores are given based on how many are identical, similar (conservative substitution) and how many gaps there are.
Can you explain the process of PCR, including the primers, taq polymerase and the three different temperatures?
1) Denaturation: Heat DNA to 98 degrees
2) Annealing: Cool to 58 dgrees to allow DNA primers to form
3) Extension: Heat it to 72 degrees AND add Taq DNA polymerase (can withstand heat)
What is a cDNA?
Copy DNA formed using reverse transcriptase.
If you have all the cDNAs that can be made from an organism, do you have its entire genome sequence?
No, does not have introns.
What is RT‐PCR? What research question is it trying to answer?
Reverse transcriptase used on isolated mRNA from a gene to get a pool of cDNA. Run PCR on gene we are interested in.
Trying to find where the gene is expressed that we are analysing.
What is qRT-PCR?
Quantitative PCR that detects the amount of light emitted
How is a Western blot run? What research question does it answer? How does it differ to an RT‐PCR?
1) Proteins heated with SDS (unfolds them) and Mercaptoethanol, making it negatively charged. Then protein is run on PAGE (polyacrylamide gel electrophoresis)
2) Transfer to a western blot (polymer sheet) where they are stained with antibodies specific to the target protein.
