Genomics

Question 1

what is a genetic map

Answer 1

diagram showing relative positions of genes on a chromosome based on how frequently they are inherited together

Question 2

how are genetic maps made

Answer 2

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

Question 3

for linked genes, what is the rate of recombination proportional to?

Answer 3

physical distance between the loci

Question 4

genetic maps vs sequencing

Answer 4

low vs high resolution to a single base pair
old vs modern

Question 5

what is the general principle of sanger sequencing/chain termination method

Answer 5

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

Question 6

how to find the complementary sequence of a section of DNA using sanger sequencing

Answer 6

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

Question 7

gel electrophoresis sanger sequencing

Answer 7

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

Question 8

how, in sanger sequencing, is it that the chain is not always terminated at same nucleotide (to produce different fragments)?

Answer 8

excess of normal dNTPs compared to ddNTPs

Question 9

what was the map based sequencing approach used by International Human Genome Sequence Consortium in the human genome project?

Answer 9

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

Question 10

what is shot gun sequencing, used by celera genomics during the human genome project?

Answer 10

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

Question 11

improvements to sangar sequencing

Answer 11

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

Question 12

removal of gels in sangar sequencing was replaced with…

Answer 12

replaced with capillary tubes to separate the fragments
detectors analyses fragments as they run through
multiple capillary tubes working in parallel
automated and efficient

Question 13

what are the steps in shotgun sequencing (4)

Answer 13

copying
fragmentation
sequencing
computational assembly

Question 14

purpose of fragmentation?

Answer 14

to create random ends that will overlap

Question 15

what is genome assembly?

Answer 15

the process of reconstructing a genome from short sequencing reads

Question 16

what are the challenges in genome assembly?

Answer 16

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

Question 17

what was left out of the human genome project?

Answer 17

centromere and telomere DNA

Question 18

what is next generation sequencing?

Answer 18

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.

Question 19

example of next generation sequencing?

Answer 19

illumina

Question 20

what is the oxford nanopore?

Answer 20

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

Question 21

pangenome

Answer 21

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

Question 22

core genome

Answer 22

genes present in all organisms of a species

Question 23

accessory genome

Answer 23

genes that vary among individuals, contributing to diversity

Question 24

why do bacteria have a wider pangenome?

Answer 24

due to plasmids and horizontal gene transfer, so multiple strains need to be screened

Question 25

meaning of annotating a genome?

Answer 25

the processing of identifying genes and their functions
involves distinguishing between coding and non coding regions and predicting where genes begin/end

Question 26

complication in eukaroytes in sequencing

Answer 26

splice variants: A single gene
can produce multiple proteins
through alternative splicing

Question 27

what is the transcriptome?

Answer 27

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

Question 28

what is transcriptomics?

Answer 28

studying which genes are turned on/off in different conditions
uses RNA sequencing and microarrays

Question 29

how does RNA sequencing work?

Answer 29

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

Question 30

what are DNA microarrays?

Answer 30

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

Question 31

what are the 2 types of fusion used in reporter gene expression assays?

Answer 31

transcriptional and translational fusion

Question 32

purpose of transcriptional fusion

Answer 32

reporter gene determines the function of a specific promoter
shows where a promoter gets turned on

Question 33

purpose of translational fusion

Answer 33

reporter gene fuses to gene of interest
shows where a protein goes in cells

Question 34

reporter genes

Answer 34

Common reporter genes include those encoding for fluorescent proteins

Question 35

green fluorescent protein

Answer 35

protein extracted from jellyfish
changing the amino acid in the central motif produced different coloured fluorescent proteins

Question 36

what is comparative genomics?

Answer 36

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

Question 37

what is the metagenome?

Answer 37

collective genetic material of microorganisms found in a specific environment.
they interact dynamically, shaping community behaviour and function
eg human microbiome