Week 1 - Tuesday 4th October Flashcards
What is the Human Genome Project?
An international research project whose mission was to decipher the chemical sequence of the complete human genetic material (genome).
Describe the process of Sanger sequencing.
- Uses oglionucleotides primers to seek out specific DNA regions.
- Begins with denaturation of double stranded DNA.
- Single stranded DNA is the annealed to oligonucleotide primers and elongated using a mixture of deoxynucleotide triphosphates (dNTP’s) which provide A, T, C and G to build the new double strand.
- A small quantity of chain terminating dideoxynucleotide triphosphates (ddNTP) is included.
- Both dNTP’s and ddNTP’s have an equal chance of attaching so chains terminate at varying lengths.
Describe the process of Next Generation Sequencing (NGS).
NGS is a new technology that is used to sequence hundreds/thousands of genes or a genome in a small period of time.
1. DNA Fragmentation - used to break DNA into short sequences eg. using sonication. The relevant DNA segments are then pulled out by hybridization capture assay. Another method involves polymerase chain reaction (PCR) amplification, which uses primers to amplify targeted DNA segents using PCR.
2. Library preparation - A process where DNA segments are modified so that each DNA segment has a sample-specific index which helps to identify the sample from whom DNA sequencing was performed.
3. Sequencing - Allows massive parallel sequencing at the same time which is analysed using bioinformatics software.
Describe the process of Nanopore sequencing.
A single molecule of DNA or RNA can be sequenced without the use of PCR amplification. Nanopore sequencing relies on the passing of single strands of DNA or RNA through a tiny protein channel (nanopore) embedded in an electircally resistant membrane. As the DNA molecule passes through the nanopore it causes a change in the ion current which can be the sequence of bases.
Describe Single Molecule, Real Time (SMRT) process.
- During library preparation, template DNA fragments are provided with hairpin structures on both sides which make the structure circular and which also provide universal binding site and initiation sequence.
- Sequencing is performed in special nanophotonic visualisation chambers referred to as zero-mode waveguide (ZMW).
- ZMW represents the lowest available volume for light detection, capable of detecting only a single nucleotide being incorporated by DNA polymerase.
- Sequencing is based on real-time imaging of distinct fluorescence labelled dNTP’s as the polymerase synthesizes DNA along single template molecules.
- SMRT technology uses four different fluorophores (one for each dNTP, so when it is cleaved in the process of replication, the signal can be measured).
- A polymerase is attached to the bottom of the chamber. The DNA loop moves through the polymerase and every time a dNTP is detained by polymerase, a phosphate bond is broken and a light pulse is produced.
- Circular DNA template allows the polymerase to continue around to the second adapter sequence.
What is the definition of ‘epigenetics’?
Epigenetics is the study of how your behaviours and environment can cause changes and affect the way your genes work.
What is the definition of DNA methylation?
DNA methylation is an epigenetic mechanism that involves the transfer of a methyl group onto the C5 position of the cytosine to form 5-methylcytosine. DNA methylation regulates gene expression by recruiting proteins involved in gene repression or by inhibiting the binding of transcription factors to DNA.
What is the definition of histone modification?
Histone modification is the addition of any chemical group to a histone which is a protein that functions to compress DNA within the nucleus to form chromatin which provides a platform for regulating gene transcription. (Acetylation, methylation, ubiquitination, phosphorylation etc.).
