The genome's content Flashcards
What four ways can chromosomes be mapped?
Karyotypic map - from microscopic observation of chromosome spreads
Linkage map - genetic map created by monitoring recombination between markers, unit is cM
Physical map - measured in base pairs, in practice may be string path of overlapping BAC clones
Sequence map - sequence of bases along chromosomes
(resolution gets higher)
Why are model organisms such as E.coli easy to sequence genomes of?
Relatively small genome size
Genetic traceability
When was the human genome project started and completed?
1990-2003
How was genetic analysis carried out before genome sequencing? (3)
Gene mapping - monitoring recombination between markers
Forward genetics - identifying mutations responsible for a phenotype
Reverse genetics - identifying phenotypes associated with a specific mutation
What do genome projects allow us to do?
Catalogue genes Facilitate forward/reverse genetics Gene expression profiling Analyse non-coding DNA sequences Compare genomes for different organisms
What is functional genomics?
Analysing gene and protein product function (and gene regulation/protein-protein interactions) using genome-wide approach with large data sets rather than on a gene by gene basis
What is comparative genomics?
Comparing genome sequence/organisation and looking at similarities and differences as well as evolutionary relationships between organisms
What does Sanger sequencing require?
Template DNA to be sequenced DNA polymerase The four DNA nucleotides A primer Dideoxy forms of all four nucleotides (ddATP, ddGTP, ddTTP and ddCTP) labelled with different colour dye
Why do dideoxy nucleotides terminate synthesis?
They lack an OH group on the 3’ carbon of the sugar ring so no further nucleotides can be added to chain
How does Sanger sequencing work?
- Primer binds template DNA and DNA polymerase adds nucleotides to chain until a dideoxy nucleotide added
- Fragments run through gel electrophoresis which separates fragments by size (short fragments move quickly, long fragments slow)
- From colours of dyes emitted the DNA sequence can be built up one nucleotide at a time
What are the limitations of Sanger sequencing?
Low throughput
Expensive
Impractical for large projects that require sequencing of large/multiple genomes
(therefore replaced by next generation sequencing technologies)
How are next-generation sequencing techniques improved?
Allow parallel sequencing of millions of DNA fragments simultaneously so increased speed and reduced costs
What is multiplexing?
Allows you to sequence millions of DNA sequences simultaneously
DNA extracted, shredded and tiled onto a surface
PCR amplifies specific sequences
Take all colonies at sequence at same time
What is ‘454’ pyrosequencing?
Shred DNA into fragments, ligate oligonucleotide adaptors and amplify by emulsion PCR
Pyrophosphate released in chain reaction and light detection acts to detect nucleotide incorporated by DNA polymerase
(454 Life Sciences unveiled first machine in 2005)
What is Illumina sequencing?
Repeated cycles of bridge amplification PCR to obtain clusters
Based on reversible dye terminators that can identify nucleotides when washed over DNA strands
