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MGD > Molecular Diagnosis > Flashcards

Molecular Diagnosis Flashcards

0
Q

Analyse nucleotide level?

A

DNA sequencing

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1
Q

What would you use to analyse DNA at gene level?

A

Restriction enzymes
DNA gel electrophoresis
PCR

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2
Q

Analyse at chromosome level?

A

Karyotyping

FISH

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3
Q

Analyse at gene level?

A

Southern hybridisation
Microarray
PCR variations

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4
Q

Analyse proteins?

A

Protein electrophoresis
Immunoassay
Enzyme Assays

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5
Q

Describe DNA sequencing?

A

Answer later

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6
Q

What are restriction enzymes?

A

Enzymes taken from bacteria that recognise specific DNA sequences as restriction sites and cut the DNA resulting in sticky ends.

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7
Q

Uses of restriction enzymes?

A

Used in fingerprinting due to DNA variation in pop
Detect mutations ie HbS
Differences in DNA fragment size ie deletion
Gene cloning ie insulin production

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8
Q

How do bacteria use RE?

A

They cut foreign DNA and degrade whereas their own DNA is protected by methylation

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9
Q

How can RE action be reversed?

A

DNA ligase is an enzyme that connects complementary sticky ends to rejoin the DNA fragments. It forms the covalent phosphodiester bonds in the sugar phosphate backbone
Important for gene cloning

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10
Q

Explain DNA electrophoresis?

A

Seperated DNA fragments by size.

  1. Solution of fragements placed in wells at the neg. anode
  2. The charge of pos. cathode encourages DNA to move towards the positive end as DNA phosphate are negative
  3. The largest fragments move the slowest hence further up the gel
  4. A DNA ladder is used as reference as are fragments of know length
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11
Q

What do you require for DNA gel electrophoresis?

A

Buffer solution
DNA fragments
power supply and electrodes
Stain to visualise the DNA fragments

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12
Q

What is gene cooing and the process of making insulin?

A

Used to make useful protein ie insulin.

  1. A plasmid is cut with REs and the gene of interest is added to a DNA vector (cut with the same RE)
  2. Using DNA ligase, this forms RECOMBINANT DNA
  3. The Recom. DNA is added to a bacterium. This is TRANSFORMATION
  4. The bacteria containing the resistant gene contains the plasmid hence is positively selected
  5. That bacteria is placed in optimal environment to multiply
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13
Q

Characteristics of plasmids?

A

Small circular DNA
Transfer to another bacteria
Can obtain anti-biotic genes

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14
Q

What is gene cloning used for?

A

Producing useful proteins
Investigate what genes do
Genetic screening
Gene therapy

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15
Q

Explain DNA sequencing?

A
  1. A flourescent/ radioactively stained ddNTPs added to DNA template strand along with DNA polymerase to create a complimentary DNA strand
  2. Depending on which ddNTP used, the DNA fragments stop at different points. Stops when ddNTP binds
  3. This produces millions of DNA fragments of different lengths
  4. They are denatured using heat and seperated by gel electrophoresis
  5. The DNA fragments are run through a computer which records the ddNTP used to recognise each base in the sequence, building from one base to millions.
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16
Q

What is the other name for DNA sequencing?

A

Sanger Dideoxy Chain Termination method

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17
Q

What is ddNTP?

A

Dideoxynucleotide tri-phosphate
Variation is dNTPs except lack a 3’ OH hence polymerisation can’t o cut and terminates the strand.

There are one for each base: ddGTP, ddATP… etc.

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18
Q

Describe PCR?

A

Amplifies DNA segments by repeating copying the target DNA using thermostable Taq DNA polymerase and pairs of primers to uniquely define a region.

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19
Q

Process of PCR?

A

Denturation - using high temp 94-96 to separate double helix

Renaturation/Annealing - lower Temp 50-65 using pair of primers

DNA synthesis- medium temperature 75-80 using Taq.

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20
Q

Uses of PCR?

A

Amplify DNA fragments specifically
Investigate single base mutations
Investigate small deletions or insertions
For DNA electrophoresis

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21
Q

Where is Taq DNA polymerase from?

A

Thermos Aquaticus

22
Q

What type of primers used in PCR?

A

Forward and reverse primers

23
Q

Describe Southern Blotting / Hybridisation?

A
  1. The unlabelled DNA separated by gel electrophoresis transferred using NYLON
  2. The fragments are then hybridised with a labelled gene (allele-specific) probe to show the specific DNA fragments
  3. Radioactive probes also mark specific complimentary DNA fragments

Eg, in normal DNA, genes for normal Hb bind but in sickle cell the probes will not bind

24
Q

Uses of Southern Blotting?

A

Investigate gene structure - ie large deletion/duplications
Investigate gene expansions and triplet codes
Investigate variation - DNA fingerprinting

25
Q

What is northern blotting?

A

RNA

26
Q

What is Western Blotting?

A

Analysis of proteins
After SDS- page
Transfer onto nylon or nitrocellulose membrane
Add primary antibodies against protein of interest
Add enzyme-linked secondary antibodies which bind to primary and produce immunoblot

Or visualised using antibody labelled to flourescence

27
Q

What are the different techniques of gel electrophoresis for proteins?

A

SDS-Page
Isoelectric Focusing
2D-Page

28
Q

What is SDS-page?

A

Proteins seperated by molecular weight.
SDS= sodium dodecyl sulphate
1. Detergent SDS denatures proteins (breaks tertiary)
2. Gives protein a neg. charge proportionate to molecular weight
3. Elctrophoresis occurs as migrate to pos. electrode.
4. Largest molecular weight (more neg.) travels further

29
Q

What is isoelectric focussing?

A

Proteins separated by isoelectric points (pI)
Proteins added to gel, with pH gradient, hence proteins migrate until they reach a pH matching their pI hence with no charge will stop migrating

30
Q

What is 2D- page?

A

Separating proteins by pI and molecular weight.
Seperate by pI, then turn gel 90* and run for molecular weight.
Important for proteomics - large scale study of proteins

31
Q

Uses of enzyme assays?

A

Measuring the activity of enzymes gives indication of heather a particular enzyme is present at normal levels.

Performed at optimal pH, temp and ionic strength as well as including appropriate ions and co-factors

32
Q

How do Enzyme Assays work?

A

Measures the production of product/ disappearance of substrate.
Continuous using spectrophotometry
Discontinuous using radioactivity or chromotography

Draw graph of the rate of enzyme activity

33
Q

Discuss proteomics?

A

It’s the analysis of all proteins expressed by genome.
Molecular diagnosis only analyses a single purified protein

Digest with trypsin
Mass spectrometry
List of peptides with sizes
Use molecular diagnosis/ list of proteins for know peptide size to identify protein

34
Q

Clinical uses of enzyme assays?

A

Metabolic disorders in tissue

Diagnosis of disease in serum

35
Q

What is ELISA?

A

Enzyme-linked Immunoabsorbent Assays
Important for serum proteins

Detects conc of protein (eg hormones) by the binding of corresponding antibodies

  1. Primary antibodies specific to protein immobilised in a solid support - microtitre well
  2. Solution to be assay applied to surface
  3. Secondary antibody conjugated with enzyme binds to antigen-antibody complex
  4. Secondary antibody binding measured by assays for enzyme activity
36
Q

What chemical marks for MI?

A

CREATINE Kinase

Lactate dehydrogenase

37
Q

Marker for pancreatitis?

A

Amylase

Lipase

38
Q

Marker for bone disorder?

A

Alkaline phophotase

39
Q

Prostate cancer marker?

A

Acid phosphotase

40
Q

What is decreased in liver damage?

A

Plasma cholinesterase

41
Q

What is inhibited in organophosphate poisoning?

A

Plasma cholinesterase

42
Q

Markers for liver damage?

A

AST & ALT

43
Q

Marker for liver damage and increased alcohol?

A

Gamma- glutamyl transferase

44
Q

Measuring blood glucose?

A

Glucose oxidase - biosensor on machines

Test strips - H2O2 converts to colour dye

45
Q

Important plasma hormones?

A 
Cortisol
T4 & T3
Insulin
TSH
Adrenaline
46
Q

How is insulin gene retrieved from human?

A

The pro insulin mRNA from human pancreas

Reverse transcriptase produces copy DNA of pro insulin which used to make recombinant DNA

47
Q

What is a heteroduplex?

A

DNA combined with a fluorescently marked probe

48
Q

Uses of reverse transcriptase?

A

Taq. Doesn’t work on RNA
Every transcribed mRNA has a polyA tail.
So use a polyT primer to make a copy of the RNA to produce cDNA using reverse transcriptase
can digest the mRNA chain therefore left with single stranded cDNA
cDNA doesn’t have interons (HENCE DIFFERENT TO GENOMIC DNA)
Can use normal PCR to amplify DNA
If high expression of gene, lots of mRNA produced therefore lots of template cDNA therefore can look at expression.

49
Q

DNA hybridisation and microarray clinically?

A

Isolate mRNA from healthy cell and tumour cell to see which genes are expressed in the two tissue types. Then put fluorescently lit probes with reverse transcriptase to produce marked cDNA (hence red is healthy and green is tumour) to see the difference in gene expression. Can see cause of tumour. Genes only expressed in healthy is red, only in tumour is green and in both is equal quantities of both. So can use a computer to see the quantities of the different colours.

Must use two conditions, healthy and diseased
Can use microarray to look at whole genome
Cells of patient compared to healthy genome, use microarray.
Extract genomic DNA from both, label with red and green then hybridised.
If you see brown, it’s in both.
If you see a red spot it means that patient has more genomic bits of DNA for that region therefore DUPLICATION
If green means that there is a bit of DNA missing therefore DELETION
Then look further to investigate which gene is specifically mutated.
Mutation that caused the disease.

Compare.

50
Q

Uses of array technology?

A

Investigate thousands of genes simultaneously
Chromosomes deletions or duplications
Conditional gene expression

51
Q

What is karyotyping?

A

Examines chromosomes in sample of cells.
Can examine the appearance and number of chromosomes and arrange in homologous pairs and order of size.
Think Down’s

52
Q

What is FISH?

A

Fluorescence in situ hybridisation

DNA. Denature and anneal a probe that is fluorescently marked

53
Q

Uses of FISH?

A

Detect genes and chromosomes in situ
- chromosomes/ gene abnormalities –> deletion/duplication/translocation

Chromosome number - painting

Chromosome behaviour - anaphase lag

MGD (5 decks)

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