genomic technologies

Question 1

What is PCR?

Answer 1

-fundamental for many DNA applications
-PCR is used to amplify a specific region of DNA
-primers flank the region you want to amplify
- Each cycle doubles the amount of DNA copies of your target sequence
increasing quantity of sample studying can help scientists carry out many experiment

Question 2

What is fragment analysis?

Answer 2

The process is used for identifying the sizes of the PCR product
-can be used to detect repeat expansions or other small size changes (up to a few hundred bp)

• PCR based assay
• PCR followed by capillary electrophoresis

Question 3

What is Huntington’s disease and what genomic technology is used to diagnose this disease?

Answer 3

• Huntingtons’s disease - severe neurodegenerative disorder
• caused by CAG repeat expansion in the HTT gene
• normal <27 copies ; intermediate 27-35 copies and pathogenic >35 copies
• expanded protein is toxic and accumulates in neurons causing cell death
• diagnosed with fragment analysis

Question 4

How is sanger sequencing of PCR product be used to determine the full nucleotide sequence?

Answer 4

• PCR products can be sequenced using sanger sequencing to get a full nucleotide sequence
• cycle sequencing : based on the same principles as PCR
• Each of the 4 DNA nucleotides has a different dye so we can determine the nucleotide sequence based on colour of the dye
• up to 800bp of sequence per reaction = good for sequencing single exons of genes
• slow, low throughput and costly to perform for large numbers of samples

Question 5

Why do we use Sanger Sequences?

Answer 5

to identify single nucleotide polymorphism(SNPs) or mutations.
R1042G mutation in gene C3 segregates with affected individuals - this mutation causes cutaneous vasculitis

Question 6

What is FISH used to detect?

Answer 6

• to detect large chromosomal abnormalities
• extra chromosomes
• Large deleted segments
• translocations

Trisomy 21 (Down syndrome)

Question 7

How does FISH work?

Answer 7

1. Design fluorescent probe to chromosomal region of interest
2. denature probe and target DNA
3. mix probe and target DNA (hybridisation)
4. Probe binds to target
5. Target fluorescences/lights up

Question 8

How is array CGH used to identify mutations?

Answer 8

array comparative genomic hybridisation

• for detection of sub-microscopic chromosomal abnormalities
• Patient DNA labelled green
• control DNA labelled red
• increased green fluorescent indicates patient DNA duplication
• increased red indicates deletion of patient DNA

Question 9

What is MLPA and what does it detect?

Answer 9

MLPA is a variation of of PCR that allows amplification of multiple targets.
Used to detect abnormal copy numbers
Used to detect submicroscopic gene deletions

Question 10

how is a complete probe made in MLPA?

Answer 10

• one probe oligonucleotide contains the sequence recognised by forward primer
• one contains the sequence recognised by reverse primer
• when both forward and reverse primers are hybridised they can be ligated to complete the probe

Question 11

What happens to the MLPA product/amplified library?

Answer 11

-perform fragment analysis
(capillary electrophoresis) of MLPA product.
-An important use of MLPA is to determine relative ploidy (how many chromosome copies?)
-Eg. probes may be designed to target various regions of chromosome of a human cell.
- The signal strengths of the probes are compared with those obtained from ref DNA sample known to have two copies of the chromosome.

Question 12

What do current strategy of next generation sequencing focus on?

Answer 12

• wider range of tests in a shorter time for less money
  Disease panels :
  -enriching to sequence only the known disease genes relevant to the phenotype
• panels expandable to include new genes as they are published
  -potentially pathogenic variants confirmed by sanger sequencing.

Question 13

what is the most common techniques in next generation sequencing?

Answer 13

• whole exome sequencing
  (~21, 000 genes in the human genome )
  -often we are only interested in protein coding exons or ‘exomes’ which makes up only 1-2% of genome
• 80% pathogenic mutations are found in exomes (protein coding)
• more efficient to only sequence the bits we are interested in, rather than the entire genome
  -cost £1,000 for a genome, but only £200 -£300 for an exome.

Question 14

How do you carry out exome sequencing?

Answer 14

• target enrichment
• capture target regions of interest with baits
• potential to capature several Mb genomic regions (typically 30 -60 Mb)

Question 15

What method has next generation sequencing replaced?

Answer 15

• Next generation sequencing has replaced sanger sequencing.

Question 16

What tests will not move to whole genome sequencing?

Answer 16

• capillary based methods : repeat expansions, MLPA, family mutation confirmation sanger sequencing.
• Array-CGH: large sized chromosomal aberrations

Question 17

What are some challenges of exome and genome sequencing?

Answer 17

1. results interpretation is the greatest challenge :
   - 20,000 genetic variants identified per coding genes ‘exome’
   - 3 million variants in a whole human genome.
2. Ethical considerations :
   - modified patient consent
   - data analysis pathways
   - strategy for reporting ‘incidental’ finding.
   - Infrastructure and training (particularly IT and clinical studies).

Question 18

What is the genome project?

Answer 18

• 100,000 genome project
• bring direct benefit of whole genome sequencing and genetics to patients
• enable new scientific discovery and medical insights
• personalised medicine

Question 19

What 3 tiers are the variants within virtual panel divided into for clinical interpretation?

Answer 19

1. tier 1 variants:
   - known pathogenic
   - protein truncating
2. tier 2 variants:
   - protein altering (missense)
   - intronic (splice site)
3. tier 3 variants:
   - loss of function variants in genes not on disease gene panel.

Question 20

What is the role of labs?

Answer 20

• main role of the lab is to help consultants reach a genetic diagnosis for individuals and families to help guide treatment and clinical management.
• perform specific tests with proven clinical validity and clinical utility
• UKGTN

Question 21

what are NHS diagnostic labs useful for?

Answer 21

1. diagnostic
2. predictive
3. carrier (recessive)
   - > diagnostic testing is available for all consultant referrals
   - > informed consent

Question 22

What are 3 diagnostic test outcomes?

Answer 22

1. pathogenic mutation
2. normal variation -polymorphism
3. novel variant -investigation to establish clinical significance

Question 23

How to establish if a mutation is pathogenic?

Answer 23

1. mode of inheritance
2. genetic databases of published and unpublished data
3. nonsense, frameshift, splice site (exon +/-2 bp) mutations
4. missense/intronic mutation - in silico tools for missense and splicing mutations

Question 24

Give an example of genetic disease case study.

Answer 24

• Mitofusin 2 (MFN2) causes charcot -Marie- Tooth disease type 2 (CMT2)
• degeneration of long nerves in legs and arms leading to muscle wasting and sensory defects.
• onset usually in childhood
• Autosomal Dominant and autosomal Recessive
• parents unaffected, siblings with severe onset CMT2
• MFN2 sequenced by next generation sequencing :
• apparently homozygous for c.647T> Cp.(Phe216ser) mutation
• Parents sequenced, expected them to both be heterozygous

Question 25

Summary : why do we do PCR and all the other sequencing methods (sanger, FISH, fragment analysis, array CGH)

Answer 25

PCR : to amplify a sample with potential disease so we can study it and carry out different sequencing methods to identify disease

Sequencing methods : all followed by PCR, the products of PCR can be used to diagnose mutations, which cause genetic disease such as Huntington’s, then use dye based methods to label and identify sequences, chromosomal abnormalities etc.