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Techniques of Molecular Medicine > Genomic technologies > Flashcards

Genomic technologies Flashcards

1
Q

What technologies do we use to analyse genomes, trandscriptomes or methylomes?

A

Microarrays

Sequencing techniques

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2
Q

Why are microarrays important for?

A

Identifying disease mechanisms

Locating which genes are switched on or off

Expression of genes following therapy

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3
Q

What are methods to carry out analysis?

A

Amplification based methods - amplify DNA or RNA (Q-PCR)

Hybridisation based methods - DNA microarrays

Sequencing based methods - next generation sequencing

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4
Q

What are the advantages of amplification based methods?

A

Extremely sensitive

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5
Q

What are the disadvantages of amplification based methods?

A

Error prone

Specific primers needed

Can be expensive

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6
Q

What are the advantages of hybridisation based methods?

A

Sensitive

Economical

Easy to analyse

High throughput

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7
Q

What are the disadvantages of hybridisation methods?

A

Limited to array content

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8
Q

What are the advantages of sequencing based methods?

A

Unlimited content

Highly parallel analyss

Base level sensitivity

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9
Q

What are the disadvantages of sequencing based methods?

A

Costly

Difficult to analyse

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10
Q

What is essential to determine before using a microarray?

A

The quality and integrity of the sample

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11
Q

What information do techniques of microarrays provide?

A

Genotyping

Methylation

Expression of genes

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12
Q

What samples can micrarrays be used on?

A

Gene

Exon

Transcriptome

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13
Q

Describe how a microarray is carried out

A
  1. Microarray chips contain mRNA sequences complementary for certain genes
  2. Scientists extract normal and abnormal cells and label them with different colours
  3. The mRNA transcribed from the genes from both cells will bind to their complementary mRNA sequences
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14
Q

How do we interpret a microarray chip?

A

If a certain gene is upregulated in the abnormal cell, the colour of the abnormal cell will predominate in that well

If a certain gene is downregulated in the abnormal cell, the colour of the normal cell will predominate in that well

If a certain gene is expressed equally in both cells, the colour of that well will be an even mixture of the two colours

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15
Q

What is microarrays used for currently?

A

Expression of genes - Studying gene expression patterns in tumours over time

Genotyping - detecting SNPs and structural variations

Methylation - detection of aberrant methylation patterns and biomarkers

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16
Q

Which cancer have been identified through microarrays?

A

Lymphoblastic leukaemia

Breast cancer

Prostate cancer

Lung cancer

Colon cancer

17
Q

Microassay principles

A
  1. Synthesised or spotted oligonulceotide/cDNA arrays on glass 50 bases in length
  2. Hybridization of RNA samples
  3. Labelling
  4. Washing
  5. Signal amplification
  6. Detection
18
Q

What is the output of expression microarrays?

A

Relative expression of genes

19
Q

What is the output of genotyping microarrays?

A

Genotype for a given probe

20
Q

What is the output for methylation microarrays?

A

Relative levels of methylation - yes or no

21
Q

Important factors to consider when designing a microarray experiment

A

Sample requirements

Quality control

Array types

Costs

Time-frames

22
Q

What are affymetrix microarrays?

A

Special microarrays designed to bind to genes, exons and transcriptomes

Contain up to 1 million different oligonulceotides, which are repeated continuously and represent known locations on the genome

23
Q

What do affymetrix microarrays allow us to do?

A

Observe which genes are upregulated or downregulated in certain conditions

24
Q

How are agilent microarrays different from affymetrix arrays?

A

You can look at the whole genome through agilent microarrays

Can only look at the exome, genome or transcriptome on affymetrix microarrays

25
Q

What is the normal processing time for microarrays?

A

Around 5 days

26
Q

What is normalisation?

A

Process of adjusting individual hybridisation intensities

27
Q

Why must data be normalised?

A

Procedures have different starting quantities of RNA

Dyes may be different at labelling

Unequal probe hybridization

28
Q

What are the 3 aims of microarray normalisation?

A

Preserving biological variation between samples

Minimising experimental variation

Allowing comparison of different experiments

29
Q

What percentage of DNA is coding?

A

0.1%

30
Q

What are GWAS?

A

Looks at many common genetic variants in different individuals to see if any variant is associated with a trait

Typically focus on the associations between SNPs and major diseases

31
Q

What is loss of heterozygosity?

A

Loss of the entire genes and the surrounding chromosomal region

Common in cancer = absence of a functional tumour suppressor gene in the lost region

32
Q

What is copy number variation?

A

A form of structural variation

Alterations of the DNA of a genome that results in the cell having an abnormal variation in the number of copies of one or more sections of the DNA

33
Q

What type of assay is illumina?

A

Genotyping assay

34
Q

What are the 4 steps to carrying out an illumina assay?

A
  1. Sample prep - adaptors added to allow the strand to bind to the complementary sequences on the lanes on the Illumina
  2. Cluster generation - sample is amplified through hybridisation and bridge synthesis
  3. Sequencing by synthesis - single fluorescent nucleotides bind to the formed sequences, releasing a fluorescent signal
  4. Data analysis
35
Q

What does the intensity of fluorescence in Illumina reveal?

A

Information about the allelic ratio

36
Q

Why are microarrays commonly used over illumina?

A

Cheap

Fast

High throughput

37
Q

What is the main disadvantage of microarray technology?

A

Cannot sequence the whole DNA sample

38
Q

Disadvantages of Illumina

A

More expensive

Low throughput

Needs bioinformatician to analyse data

Techniques of Molecular Medicine (12 decks)

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