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Health and Disease: Genes > Blair Genetic disease > Flashcards

Blair Genetic disease Flashcards

1
Q

How big is the human genome?

A

3300mb/ 21,000 nuclear genes

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2
Q

How many mitochondrial genes?

A

37 genes, 16.6kb

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3
Q

What proportion of the genome is exonic?

A

2.5%

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4
Q

What does extragenic DNA consist of?

A

mostly repetitive sequences

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5
Q

What length of extragenic repeat is significant to implicate disease?

A

1x10^5

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6
Q

What are the 2 types of DNA repeat?

A

tandem and interspersed

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7
Q

What is polymorphic DNA?

A

DNA that varies between individuals

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8
Q

What are the classes of tandem DNA repeat?

A

satellite repeats, microsatellites, minisatellites

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9
Q

Where do minisatellites exist?

A

telomeres

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10
Q

Where do satellites exist?

A

heterochromatin

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11
Q

What size are microsatellites?

A

mostly dinucleotides (STRs)

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12
Q

Where are microsatellites found?

A

All chromosomes

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13
Q

What are VNTRs?

A

Variable number tandem repeats- minisatellites

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14
Q

What are microsatellites also known as?

A

STRs

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15
Q

Which type of tandem repeat has most repeats?

A

STR

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16
Q

What can polymorphisms be used to detect?

A

Paternity, forensics, inheritable disease

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17
Q

How are VNTRs detected?

A

restriction fragment polymorphisms change length in electrophoresis and southern blotting. Multi-locus probes detects many related sequences of different lengths. Single Locus Probes detect adjacent sequences for high specificity- sequencing for copy number

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18
Q

What are the failures of SLP probing?

A

Allele drop out caused by PCR of low copy numbers, easily contaminated

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19
Q

What are SNPs?

A

Single nucelotide polymorphisms.

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20
Q

Mismatch probes will not anneal in high stringency conditions…

A

T close to Tm, low salt

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21
Q

How are SNPs directly detected?

A

Oligonucleotide Ligation Assay, Amplification Refractory Mutation System, Single Stranded Chain Polymorphism.

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22
Q

How does OLA work?

A

2 probes, common probe fluoresces to detect with capillary electrophoresis. Ligates if mutant probe has 3’ complementary base. Varying length Polyethoxy tail for normal and mutant

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23
Q

Which detection of SNPs requires a known mutation?

A

OLA ARMS

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24
Q

How does ARMS work?

A

Primer has 3’ mutant and extension only if mutant present due to altered Tm

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25
Q

Does SSCP require a known SNP?

A

No

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26
Q

How does SSCP work?

A

PCR amplification. Secondary structure lost after heating and cooling to increase PAGE migration

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27
Q

What techniques are used in clinical diagnostics?

A

high throughput: capillary electrophoresis, fluorescence/chemiluminescence, solid phase capture

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28
Q

What are the high throughput methods used to detect sequences?

A

Taqman, SybrGreen, Lux, qPCR, molecular beacons, scorpion probes, sp FRET, DGGE, gene chips

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29
Q

How does Taqman probes work?

A

Fluoresce when displaced by Pol 2

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30
Q

How does qPCR work?

A

fluorescent nucleotides extend proportional to starting DNA concentration

31
Q

How do Lux probes work?

A

Fluoresce when extended

32
Q

What does SybrGreen bind?

A

non specific for dsDNA

33
Q

How do molecular beacons work?

A

annealing releases quencher for fluorescence

34
Q

How do scorpion probes work?

A

Molecular beacon attached to PCR primer. amplification causes self annealing and releases quencher

35
Q

How does spFRET work?

A

OLA with 2 fluorophores, ligation, denaturation, produces secondary structure for FRET

36
Q

How does DGGE work?

A

Progressive denaturation of discrete domains produces single strand which anneals to gel. separates oligonucleotides, small DNA fragments, PCR products

37
Q

How are gene chips synthesised?

A

3’-5’ on using light to block addition of nucleotides. Can be made to any sequence.

38
Q

What do microarrays do?

A

different regions for each mutant, fragment annealing can be detected for complementary sequence

39
Q

Why is sequencing not used?

A

expensive to sequence whole genome

40
Q

When is sequencing used?

A

for small fragments, detected by RNA bait on strepatividin beads

41
Q

What is first generation sequencing?

A

Sanger/dieoxy NTP sequencing uses termination, electrophoresis then colour reading

42
Q

What are the types of second generation sequencing?

A

Illumina, Roche, ABI

43
Q

How does Illumina sequencing work?

A

fluorescent reversible terminators

44
Q

How does Roche sequencing work?

A

PCR amplification releases PPi for ATP for luciferase. one base at a time.

45
Q

How does ABI sequencing work?

A

Fragment bound to bead with P1 primer tag. Mutating primer gives sequence by moving along fragment

46
Q

What is gene therapy?

A

Deliberate introduction of nucleic acids into human somatic cells for therapeutic, prophalatic or diagnostic purposes

47
Q

What does gene therapy treat?

A

Single locus gene defects, eradicate tumour growth, stimulate immune response or control autoimmune disease

48
Q

Why are vectors needed for gene therapy?

A

Cells are resistant to free nucleic acids. Targetting of specific cell types.

49
Q

What are the 2 main types of Gene therapy vectors?

A

liposomes and viruses

50
Q

How do liposomes bind DNA?

A

Phosphates bind on outside of weakly cationic phospholipids.

51
Q

What are the advantages of liposomes?

A

No immunological response allows multiple treatments. Can administer large genes.

52
Q

What are the disadvantages of liposomes?

A

Poor targetting, poor uptake and poor delivery into the nucleus. Epichromosome is less stable.

53
Q

What are the advantages of viral vectors?

A

Efficient targetting and entry

54
Q

What are the 3 modes of viral DNA containing?

A

Sustained integrated; Non-integrating transient; non-integrating sustained

55
Q

Which type of DNA incorporation in viral vectors is most common?

A

integrating

56
Q

What are the disadvantages of viral vectors?

A

Produce inflammation. Transient requires multiple administrations but immune response. Integrating can disrupt host genes. Expensive to produce replication deficient species.

57
Q

What are the integrating viral vectors?

A

reteroviruses and adeno-associated viruses

58
Q

How do reteroviruses carry genes for therapy?

A

RNA for reverse transcriptase inserted into dividing cells

59
Q

Why are reteroviruses replication deficient?

A

No PS1 coat protein. (produced in packaging cells)

60
Q

When do adeno-associated viruses become active?

A

Insert ssDNA into Chromosome 19 in presence of adenovirus.

61
Q

What type of viral vector is Herpes?

A

A sustained, non-integrating

62
Q

Why is herpes virus used?

A

Many non-essential genes, latent infection established in non dividing cells evades immune response

63
Q

What do adeno-associated viruses treat?

A

occular disease

64
Q

What do herpes virus treat?

A

Neurological disease

65
Q

What type of vector is Adenovirus?

A

An integrating viral vector

66
Q

What cells does adenovirus target?

A

non-dividing epithelial cells (51 total targets)

67
Q

Which genes of adenovirus are used as vector?

A

E1A/B for growth and activation of packaging late genes, E3. Replacement with insert up to 10kb.

68
Q

How is adenovirus produced?

A

E1A constitutively expressed in 293 packaging cells

69
Q

How does adenovirus target cells?

A

fibre structure within capsid: CAR receptor D1 domain bound by knob, penton RGD motif binds integrins.

70
Q

How has adenovirus been modified for second generation therapies?

A

ONYX-015 targets p53 deficient cells,
E2 DNA binding protein replacement is replication defiecient,
“gut” viruses have only terminal repeats requiring cotransfection
modification of fibre for novel receptors

71
Q

How is gene therapy administered in vivo?

A

intravenous, topical, direct, nebulisation

72
Q

Which disorder has been treated by ex vivo gene therapy?

A

SCID treated with reterovirus using cytokine receptor subunit, but random integration upregulated transcription factors causing leukaemia.

73
Q

What barriers are there for adenovirus?

A

CAR in tight junctions (growth inhibitor), loss of CAR (tumour supressor) in cancer, immune response

74
Q

Why is lentivirus a better viral vector?

A

Reterovirus can target non dividing cells, 3 genes for simple modification

Health and Disease: Genes (6 decks)

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