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MGD > Molecular Diagnosis > Flashcards

Molecular Diagnosis Flashcards

1
Q

Describe DNA sequencing using the Sanger Dideoxy Chain Termination method

A

Fluorescent ddNTPs and regular dNTPs added to a DNA template strand along with DNA polymerase - complementary DNA strand. Strand terminates, depending on strand. Produces lots of new DNA fragments of different lengths which can be denatured and separated by gel electrophoresis.

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2
Q

Why do ddNTPs cause termination?

A

They lack an -OH group so polymerisation cannot occur

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3
Q

What can restriction analysis be used for?

A

Size of DNA fragments, mutations, DNA variation, gene cloning

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4
Q

Describe gene cloning

A

Plasmid is cut using restriction enzymes, gene of interest is added to create recombinant DNA molecule, introduced into bacterium, placed in an environment to multiply

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5
Q

What are plasmids?

A

Small, circular DNA which can transfer from one bacterium to another. They can contain antibiotic genes

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6
Q

Describe DNA gel electrophoresis

A

Solution of different fragments is placed in a well at the cathode end (-ve), charge encourages DNA to move towards anode, larger fragments move slowest. Fragments of known size are used as reference

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7
Q

What does gel electrophoresis require?

A

Gel, buffer, power supply, stain

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8
Q

What does PCR do?

A

Amplifies DNA segments by repeated copying of target DNA using thermo-stable DNA polymerase (Taq polymerase) and primers. Exponential growth

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9
Q

Describe the process of PCR

A

Denaturation (95°C), rennaturation (annealing of primers to DNA) at 55°C, DNA synthesis by Taq polymerase at 72°C. 5’–>3’ synthesis.

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10
Q

What is PCR used to investigate?

A

Single base mutations, small deletions or insertions

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11
Q

Describe southern blotting/hybridisation

A

Nylon is used to transfer fragments from gel electrophoresis, hybridised with labelled gene probes to show specific DNA fragments. Radioactive probes - mark specific complementary DNA fragments

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12
Q

What is the technique southern blotting used to investigate?

A

Gene structure (large deletions/duplications), gene expansions (triplet repeats e.g. fragile X syndrome) and variation (e.g. DNA fingerprinting)

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13
Q

Describe northern blotting

A

Nylon is used to transfer fragments from gel electrophoresis, hybridised with labelled gene probes to show specific RNA fragments. Radioactive probes - mark specific complementary RNA fragments

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14
Q

Describe western blotting

A

Nitrocellulose membrane is used to transfer fragments from gel electrophoresis, specific proteins can be visualised using antibodies conjugated to a label

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15
Q

How can PCR be used in an allele-specific test?

A

Use primers to sequence either side of allele of interest in order to amplify that specific allele

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16
Q

How can restriction analysis be used in an allele-specific test?

A

Use one/multiple restriction enzymes with restriction sites around/within the allele. Analyse size of fragments produced. If wild type is cut but not the sample then the restriction site is mutated/missing

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17
Q

How can DNA hybridisation be used in an allele-specific test?

A

Use a DNA probe that is complementary to either the wild type allele or mutated allele, see which binds

18
Q

Describe SDS-Page

A

Detergent SDS denatures proteins and binds to give them a -ve charge proportionate to Mr. Then do electrophoresis, largest Mr moves most (most -ve)

19
Q

Describe isoelectric focusing

A

Proteins applied to gel containing a pH gradient. Protein will migrate until it reaches a pH that matches it’s pI - no overall net charge

20
Q

What basis does SDS page separate proteins on?

A

Molecular weight

21
Q

What basis does isoelectric focusing separate proteins on?

A

Charge

22
Q

Describe 2D-page

A

Gel is turned by 90° and run for a different property

23
Q

Why is 2D page useful?

A

Allows separation of proteins that have an identical pI but different Mr’s (or vice versa). Important for proteomics

24
Q

What conditions are needed for enzyme assays?

A

Optimal pH, temperature and ionic strength. Appropriate ions or cofactors for enzyme action

25
Q

What is measured in an enzyme assay?

A

Production of product or the disappearance of substrates

26
Q

What enzymes can be markers for liver damage/disease?

A

Aspartate transaminase (AST), alanine transaminase (ALT)

27
Q

What enzyme is a marker for myocardial infarction?

A

Creatine kinase

28
Q

Which enzymes are markers for pancreatitis?

A

Lactate dehydrogenase, amylase, lipase

29
Q

What is gamma glutamyl transferase a marker for?

A

Liver damage, increased by alcohol.

30
Q

What is alkaline phosphatase a marker for?

A

Bone disorders

31
Q

What is a marker for prostrate cancer?

A

Acid phosphatase (ACP)

32
Q

What is plasma cholinesterase a marker for?

A

Decreased levels in liver disease, inhibited in organophosphate poisoning

33
Q

Describe enzyme-linked immunoabsorbent assays (ELISA)

A

Primary antibody is immobilised on a solid support, solution to be assayed is applied, secondary antibody conjugated with an enzyme binds to antibody-antigen complex, binding of second antibody is measured by assaying

34
Q

What are ELISAs used for?

A

Detection of the concentration of a protein by the binding of its corresponding antibody

35
Q

What are arrays used to investigate?

A

Sub-microscopic chromosomal deletions.

36
Q

Describe an array

A

DNA probes of entire genome is applied to surface of a solid matrix, patient and normal DNA labelled differently, equal amounts of both are hybridised to the probe array and signals are detected, for probes where signal of normal DNA exceeds that of patient DNA the patient has a deletion

37
Q

What is FISH used for?

A

Used to detect and localise the presence (or absence) of specific DNA/RNA sequences on chromosomes

38
Q

Describe fluorescent in situ hybridisation (FISH)

A

Chromosomes inside cells, fluorescent probes used for a specific gene or chromosome, seen with fluorescence microscopy

39
Q

Describe chromosome painting

A

Uses fluorescently labelled DNA probes which are hybridised to chromosomal DNA. Hybridisation - produces a fluorescent display which can be used

40
Q

What can chromosome painting be used for?

A

Genes in situ, chromosome number, chromosome structure, chromosome behaviour

41
Q

Describe karyotyping

A

Collect cells undergoing mitosis, suspend them, stain cells, take digital photographs and rearrange them electronically

42
Q

Why would you perform an allele-specific test?

A

Detection of mutations, restriction enzyme site, sequence of DNA

MGD (11 decks)

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