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Intercal - Advanced molecules, membranes and cells > Genomic Analysis > Flashcards

Genomic Analysis Flashcards

1
Q

Define genomics

A

The study of all the nucleotide sequences including structural genes, regulatory sequences and non-coding DNA segments in the chromosomes of an organism

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2
Q

What part of the protein cycle does genomics look at

A

Just DNA

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3
Q

What part of the protein cycle does functional genomics look at

A

The characterization of protein-DNA interactions on the genome of an organism

Looks at - DNA, RNA, Proteins

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4
Q

Define structural genomics

A

the dissection of the architectural features of genes and chromosomes - how they’re packaged up and their location

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5
Q

Define comparative genomics

A

the evolutionary relationships between the genes and proteins of different species

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6
Q

What does epigenomics/epigenetics look at

A

DNA methylation patterns, imprinting and DNA packaging

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7
Q

Define pharmacogenomics

A

new biological targets and new ways to design drugs and vaccines using genes.
E.g viral knock in etc

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8
Q

What is a genome

A

The single nucleotide sequence of an organisms hereditary information (DNA in humans).

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9
Q

How many base pairs are in the human genome

A

3.2X10[11] -3.2 billion

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10
Q

What was the first RNA genome sequenced

A

Bacteriophage MS2 - 1976

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11
Q

What was the first DNA genome to be sequenced

A

Phage Phi-X174 - 1977

Small with only 11 genes - this is to get in and out infected cells ASAP (smash and grab approach).

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12
Q

What is the trend between genome size and the number of genes

A

There is no real trend

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13
Q

What 3 things did the human genome project discover

A

Large centromeres of unsequenced repetitive data

21,700 genes

Only 1.5% actually code for proteins, the lowest % of all organisms

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14
Q

Of the 98.5% of the genome that don’t code for proteins, what does the rest do

A

Introns
Regulatory sequences (promoters etc)
Unique non-coding DNA
Repetitive DNA

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15
Q

How do mutations help calculate the age of an organism and its divergence from the evolutionary tree

A

DNA incorporates mutations at roughly an equal rate - 10[-5]-10[-6] mutations per base pair per generation.

This can act as a molecular clock - more mutations means more divergence from a common ancestor therefore the “newer” the species. (e.g. humans have more mutations than dinosaurs).

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16
Q

How do you compare genomes

A

Use a genome browser - can compare many species DNA sequences with each other
Computer algorithms align the sequences and provide a visual output of how alike they are.

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17
Q

What can genome browsers help show

A

Areas of the genome that are conserved throughout time across species - meaning the regions that are highly similar must be important to survival (Evolutionary conserved regions -ECR)

These similar sites are assessed to look for transcription factor binding sites.

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18
Q

Why is functional genomics, the study of DNA-protein interaction, important

A

Mis-regulation of transcription factors will change gene activity (up/down/on or off) and ultimately lead to changed protein levels = disease

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19
Q

How is a gene turned on (gene regulation)

A

Dependent on surface receptors, these allow substrates to bind which triggers intracellular pathways to recruit RNA polymerase by binding to the enhancer and start transcription and translation to make a protein from the desired gene.

20
Q

What is the role of an enhancer

A

Fine tunes the expression of a gene by turning its transcription activity up and down, dependent on the proteins that bind to that receptor

21
Q

What is the role of a promoter

A

Turns gene on

22
Q

How does DNA footprinting work (used for finding transcription factor binding sites)

A

Label a short sequence with a radiolabel/florescent label (32p) with the transcription factor on this sequence.

Mix these sequences with transcription factors

Cleave this mixed sample where it shall cut around the transcription factor due to not being able to get into cut it because it is bound to the transcription factor.

Control - Cut up the same bit of DNA without a transcription factor, will now be cut randomly and through the desired gene

Carry out gel electrophoresis - these fragments form a ladder based on size. Missing area where the gene should be (showing that the transcription factors do bind to your desired gene).

23
Q

How does chIP seq work (chromatin immunoprecipitation sequencing) - used for finding transcription factor binding sites

A

Find binding sites using genetic browser (enhancer and promoter usually).

Treat with formaldehyde to fix tissue (covalent link between everything in the cell).

Ultrasonic waves to break open cells and smash the DNA into small fragments (500-1000BP)

Add antibody to bind to protein of interest

Magnetic beads coated in specific protein for antibody are fished out with magnet.

Wash beads (with antibody, transcription factor and DNA) to remove everything else

DNA is purified with chloroform extraction and used.

Pure DNA sample that used to be bound to TF is then sequenced.

Gives you billions of 50 base pair sequences

Computers take these genome parts and align them with human genome

Because antibodies have been used to find the DNA originally, these DNA sequences will be much more common than the other non-attached parts of DNA in that gene.

This large quantity of attachment DNA sections show the areas where TFs bind in the gene.

24
Q

What two factors are vital for ChIP seq

A

Must have a completed genome as a reference to start with and reliable antibodies to bind to desired protein to fish it out.

25
Q

Why is chIP seq good

A

Can look in whole genome
Can see transcription factor working and therefore helps to determine if it is affected by the genes activation process

26
Q

Define epigenetics

A

The study of heritable changes in gene function that can occur without the DNA sequence changing

27
Q

Give 3 examples of epigenetics

A

DNA methylation – can activate or repress regions of genome

Chromatin remodeling – euchromatin (open) and heterochromatin (closed) caused by histone changes

Gene silencing – through the above mechanisms

28
Q

How can methylation be inherited

A

Methyltransferase enzyme only adds methyl group to C when in CG sequence that is opposite an already methylated site on the opposite strand.

29
Q

How can changes to chromatin be inherited?

A

The heterochromatin proteins are bound to the histones. It unbinds to allow chromosome duplication and in the two new daughter cells it rebinds in the same area.

30
Q

How can epigenetics be linked to disease

A

Accidental packaging of DNA into hetrochromatin so isnt transcribed and translated in that specific cell type and its daughter cells, meaning the protein isnt produced.

Methylation of a cytosine residue when it shouldn’t can silence or activate genes

31
Q

How does epigenetics differ for genetic mutations that cause disease

A

Epigenetics - usually individuals

Genetics - usually population (CF)

32
Q

What is the aim of pharmacogenomics and why

A

Tailored drugs to an individual to give more effective treatments

Every patient will metabolise drugs differently, so understanding genetic differences may help to overcome problems with patients being unresponsive to certain treatments

33
Q

Give an example of how asthma can be treated with pharmacogenetics

A

Multifactoral so hard to do maintenance treatment.
Can help to treat based on targeting their specific genomes e.g. ADRB2 gene

34
Q

What can DNA microarrays do for pharmacogenetics

A

Can give genome wide expression profiles to see what genes are over expressed and under expressed in specific diseases

35
Q

How do microarrays work

A

Put a normal cDNA sample from undiseased person and diseased cDNA sample from diseased person and carry out RT-PCR (reverse PCR) to form DNA and then carry out PCR to amplify it.

Label the amplified DNA with florescent dyes (green - normal and red - diseased).

Mix them in equal amounts to create a hybrid (yellow).

Scan -
- more red - over-expression of diseased gene
- more green - under-expression of diseased gene (sliding scale so can give a quantity of imbalance).

This does this for all the genes in the human genome - allowing us to look at over/under-expression of any gene for a person.

36
Q

How is genome-wide association study (GWAS) used to identify diseases

A

Look for single nucleotide polymorphisms (SNPs) within the genes that code for the proteins.

Can be used to tailor medicine to treat disease.

SNPs are just different alleles not necessarily mutations

37
Q

What is the future of genomics

Wider reading - gibson 2010

A

Trying to remove genome from one organism yeast and replace it with M. mycoides genome (bacteria).

This process, if successful, would allow easy manipulation of its genome to do various things - produce oil, clean up oil, produce drugs

38
Q

How are these bacterial cells with the chemically synthesized genome made

A

1078bp cassettes were overlapped by synthetic oligonucleotides into sets of 10 to produce 10Kbp assembilies.

These 10Kbp were then combined into sets of 100kbp.

These 10Kbp were combined with another 9 to form a plasmid for yeast implantation.

39
Q

What did comparitive genomics discover regarding motif regions

WIDER READING - Xie 2005

A

Motif regions - short, recurring patterns in DNA that are presumed to have a biological function. Often they indicate sequence-specific binding sites for proteins such as nucleases and transcription factors (6-10bp)

They carried out comparative analysis of humans, mice rat and dogs genomes and found 105 new motid sites likely to be involved in post-transcriptional regulation - half code for microRNA (non-coding RNA that binds to 3 end and regulates gene expression and many mRNAs by degredation of them. Meaning that microRNA may be more abundant than previously believed.

40
Q

What role does epigenetics have in cancer

WIDER READING - Morales ruiz

A

Cancer cells show abnormal DNA methylation patterns. Cancer is marked by global hypermethylation, which destabilizes the genome and activates oncogenes. Paradoxically, it is also marked by hypermethylation at specific sites. This turns off tumor suppressor genes. One goal of epigenetics, therefore, is to restore normal DNA methylation patterns.

41
Q

How did they reverse methylation in colorectal cancer cell

WIDER READING - Morales ruiz

A

Expression of plant 5mC DNA glycosylase induces genome-wide changes in the methylome of CRC cells and important alterations of their phenotype.
Can bind these plant domains with a DNA binding domain to work in humans
Have to use plant as no human alternatives

42
Q

ChIP seq in identifying heart enhancers

WIDER READING - blow et al

A

Lack of enhancers identified in heart tissue compared to other tissue types.

Used mice and Chip seq to identify 3000 possible enhancers with P300 binding (transcriptional co-activator) which showed 130 of these that were tested were active. This was compared to midbrain enhancers which showed that heart enhancers were overall, poorly evolutionary conserved.

43
Q

How was comparative genomics used in identifying pathogen-resistant rapeseed

WIDER READING

A

Found nucleotide-binding leucine-rich repeat (LRR) receptors (NLRs) control resistance against intracellular (cell-penetrating) pathogens in various locus
Resistance to pathogens -

NLR genes in resistance against the intracellular pathogen P. brassicae and a putative NLR gene in Rlm9-mediated resistance against the extracellular pathogen L. maculans.

44
Q

What did pharmacogenetics discover regarding abacavir hypersensitivity (HIV patients)

WIDER READING - Mallal 2002

A

57.1 ancestral haplotype causes abacavir hypersensitivity in this with HLA b5701 gene.

If abacavir is withheld it should reduce hypersensitivity from 9% to 2.5% without denying abacavir to any patients.

45
Q

What did large scale genomic analysis discover regarding CVD

WIDER READING Schunkert et al

A

13 new susceptible loci for CVD -

ABO and ADAMTS7 - Atherosclerosis

CNNM2 - hypertension

Intercal - Advanced molecules, membranes and cells (31 decks)

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