Genomics Flashcards
what is a genetic map
diagram showing relative positions of genes on a chromosome based on how frequently they are inherited together
how are genetic maps made
analyse cross over frequency during meiosis- tells us how far apart two genes are on a chromosome
genes that are closer together will have a low cross over frequency and are more tightly linked
for linked genes, what is the rate of recombination proportional to?
physical distance between the loci
genetic maps vs sequencing
low vs high resolution to a single base pair
old vs modern
what is the general principle of sanger sequencing/chain termination method
incorporation of chain-terminating dideoxynucleotides (ddNTPs) by DNA polymerase during DNA replication. These modified nucleotides lack a 3’-OH group, which prevents the addition of further nucleotides, effectively terminating the DNA strand
how to find the complementary sequence of a section of DNA using sanger sequencing
attach a primer to it that will extend when DNA polymerase added
can get the extension to stop at a known nucleotide base, but it can be done so that it does not stop everytime
perform gel electrophoresis
gel electrophoresis sanger sequencing
smaller fragments run further through the gel
used radiolabelled primers so that x ray film can be used to determine the sequences of the DNA fragments
you read it from the bottom-up to get the sequence
how, in sanger sequencing, is it that the chain is not always terminated at same nucleotide (to produce different fragments)?
excess of normal dNTPs compared to ddNTPs
what was the map based sequencing approach used by International Human Genome Sequence Consortium in the human genome project?
creates a map of the genome then divides it into large fragments before arranging them into an order based on known genetic markers
each fragment was sequenced individually then assembled in order
what is shot gun sequencing, used by celera genomics during the human genome project?
randomly breaking the genome into many small fragments, sequencing these fragments, and then using computational methods to reassemble the original sequence by finding overlaps between the fragments.
used for sequencing large genomes
improvements to sangar sequencing
used fluorescent probes rather than radiolabelled to enable quicker analysis
could all be run on the same gel as different colours
could run different lanes to improve efficiency
removal of gels in sangar sequencing was replaced with…
replaced with capillary tubes to separate the fragments
detectors analyses fragments as they run through
multiple capillary tubes working in parallel
automated and efficient
what are the steps in shotgun sequencing (4)
copying
fragmentation
sequencing
computational assembly
purpose of fragmentation?
to create random ends that will overlap
what is genome assembly?
the process of reconstructing a genome from short sequencing reads
what are the challenges in genome assembly?
No sequencing is entirely accurate, so each region needs to be sequenced multiple times to identify errors.
repetitive sequences (transposons, tandem repeats, centromeres) are difficult to sequence and can lead to errors
requires a lot of computer power
what was left out of the human genome project?
centromere and telomere DNA
what is next generation sequencing?
sequences millions of fragments of DNA or RNA in parallel, which significantly speeds up the process compared to traditional methods like Sanger sequencing. This technology can generate vast amounts of data in a relatively short amount of time.
example of next generation sequencing?
illumina
what is the oxford nanopore?
a small protein embedded in an artificial membrane that allow DNA/RNA strands to pass through while the system measures changes in electrical current
can do very long reads
can sequence repetitive sequences
high error rate (5-15%) due to the pore not being inhabited by a single nucleotide (about 5)
completed the human genome project
pangenome
The complete set of genes within a species, encompassing both core genes shared by all individuals and variable genes present in some but not others
core genome
genes present in all organisms of a species
accessory genome
genes that vary among individuals, contributing to diversity
why do bacteria have a wider pangenome?
due to plasmids and horizontal gene transfer, so multiple strains need to be screened
meaning of annotating a genome?
the processing of identifying genes and their functions
involves distinguishing between coding and non coding regions and predicting where genes begin/end
complication in eukaroytes in sequencing
splice variants: A single gene
can produce multiple proteins
through alternative splicing
what is the transcriptome?
all of the RNA molecules transcribed from a genome
it is dynamic and constantly changing in response to the cells environment/external stimuli.
all types of RNA. they are transcribed from active genes so reflect the cell’s current gene expression profile
what is transcriptomics?
studying which genes are turned on/off in different conditions
uses RNA sequencing and microarrays
how does RNA sequencing work?
The mRNA is isolated using poly(T) beads Poly T primers bind to polyA tails that bind to the beads. mRNA isolated from cell lysates and then converted to cDNA by reverse transcriptase. cDNA sequenced to measure gene expression levels and see which are active
what are DNA microarrays?
small chips used to simultaneously detect expression of many genes at once by measuring the level of mRNA in a sample, providing a snapshot of which genes are expressed under certain conditions
each spot contains a unique DNA sequence corresponding to a specific gene. the RNA is reverse transcribed into the cDNA and labelled with a fluorescent probe. the cDNA then binds to its complementary DNA on the array. the intensity of the signal from each spot shows how much mRNA is present for that gene, giving an idea of the expression level
what are the 2 types of fusion used in reporter gene expression assays?
transcriptional and translational fusion
purpose of transcriptional fusion
reporter gene determines the function of a specific promoter
shows where a promoter gets turned on
purpose of translational fusion
reporter gene fuses to gene of interest
shows where a protein goes in cells
reporter genes
Common reporter genes include those encoding for fluorescent proteins
green fluorescent protein
protein extracted from jellyfish
changing the amino acid in the central motif produced different coloured fluorescent proteins
what is comparative genomics?
comparison of genomes across species to reveal evolutionary relationships
pinpoint genetic variations that may explain why certain species have unique traits
used to determine mutations in the genome
what is the metagenome?
collective genetic material of microorganisms found in a specific environment.
they interact dynamically, shaping community behaviour and function
eg human microbiome
