Genome Projects Flashcards
What is a genome?
Entire genes/All the DNA of an organism in the nucleus a cell.
How does a genome project work?
It works by collecting DNA samples from many individuals of a species. These DNA samples are then sequenced and compared to create a reference genome.
What is the human genome project?
- HGP began in 1990 as an international, collaborative research programme.
- Publicly funded
- DNA samples were taken from multiple people around the world, sequenced and used to create a reference genome.
- Data made publicly available
What is a proteome?
All the proteins made by an organism.
Why is determining the proteome of humans difficult?
Contains large amounts of non-coding DNA which are hard to distinguish from coding DNA.
What does DNA sequencing allow?
The base sequence of an organism’s genetic material can be identified and recorded.
Most DNA sequencing is now…
automated.
What nucleotides are used in DNA sequencing?
Dideoxyribose
What is automated DNA sequencing?
- makes use of the chain-termination technique
1. A short length of DNA is chosen and inserted into a vector as a single strand of DNA.
2. A primer is annealed (hybridised) to the start of the recombinant DNA.
3. During the incubation period, DNA polymerase is added to the recombinant DNA alongside a mixture of deoxyribose nucleotides containing all 4 bases.
4. Usually, DNA polymerase attaches to the primer and begins DNA replication of the single strand recombinant DNA.
5. Hydrogen bonds form between the complementary bases on deoxyribose nucleotides. However, a mixture of dideoxyribose nucleotides are also present.
6. DNA polymerase can insert one of the dideoxynucleotides by chance which results in the termination of DNA replication.
7. When there is a sufficient ratio of deoxynucleotides to dideoxynucleotides complementary, DNA chains of varying lengths are produced.
8. Each type of dideoxynucleotide is labelled using a specific fluorescent dye:
A = green
T = red
C = blue
G = yellow
9. Once the incubation period has ended and the dideoxynucleotides have bound to their complementary bases the DNA chains are removed from the template DNA.
10. The single-stranded DNA chains are then separated according to size using a specific type of electrophoresis that takes place inside a capillary tube. - An automated DNA sequencing machine can read roughly 100 different DNA sequences within 2 hours.
What is manual DNA sequencing?
- A separate run is required for each type of dideoxynucleotide - ddNA, ddNT, ddNC and ddNG
- The dideoxynucleotides are labelled using radioactivity instead of fluorescent dyes
- After the incubation period, the four separate mixtures are added to separate wells in a gel and separated using gel electrophoresis
- A Southern transfer is made using the electrophoresis gel and an autoradiograph is taken of the Southern transfer
How are results from manual DNA sequencing interpreted?
- Autoradiograph
- Below each well there is a track of bands produced from DNA replication in the presence of each type of dideoxynucleotide
- The band that moves the furthest is the smallest DNA fragment
- The smallest DNA fragment that can be formed from the chain termination technique is one nucleotide long so is the first base in the sequence of the developing strand, and so on
Why is it easier to sequence genome from prokaryotic than eukaryotic
- Prokaryotic organisms do not contain introns in their DNA
- So genome can be used directly to sequence the proteins directly that derive from genetic code
Why cant the genome be easily used to translate the proteome in eukaryotic cells?
- introns: base sequences that don’t code for amino acids therefore proteins
- regulatory genes: STOP & START codons as they don’t directly code for amino acid
Why is the proteome larger than the genome?
- Alternative splicing: removal of introns & re-joining of introns in different combinations to create collection of mRNA w/ different functions
- Modification of proteins (in Golgi apparatus)
