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MBBS IV - Supplementary Courses > Molecular Genetics - Techniques for genetic analysis (I) > Flashcards

Molecular Genetics - Techniques for genetic analysis (I) Flashcards

1
Q

Define molecular pathology

A

Investigation and understanding of origin and mechanism of human diseases at the molecular level

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2
Q

Application of molecular pathology in Oncology

A
  • Develop molecular approaches for Dx and Classification of human tumors
  • Design and validate Predictive Biomarkers for disease progression
  • Maximize response to treatment
  • Genetic susceptibility to develop cancer
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3
Q

Give one example of cancer molecular prognostic marker and method of testing

A

BCR/ABL kinase mutation for Chronic myeloid leukemia (CML) and related disorders (e.g. ALL)

cDNA extracted from peripheral blood > semi-nested PCR and sequencing > find t(9:22) bcr/abl fusion

Application: BCR/ABL gene fusion = use specific tyrosine kinase inhibitors/ Mutation in ABL domain = use alternative inhibitors

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4
Q

Give one example of molecular pathology test for viruses and application

A

HPV

Test: PCR-based test to detect high risk HPV 16,18 subtypes

Application: Screen for cervical cancer, monitor disease recurrence

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5
Q

Give one example of molecular pathology test for hereditary cancer and disorders

A

BRCA 1/2 mutation screening for breast or ovarian cancer

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6
Q

Give one example of molecular pathology test for toxicology screening

A

Predict adverse drug reaction

e.g. Carbemazepine need HLA typing

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7
Q

Define the specific tubes required for routine collection of whole blood, plasma and serum

A

Whole blood = EDTA blood tube or clotted blood tube
Plasma = EDTA blood tube
Serum = Clotted blood tube

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8
Q

Difference in collecting whole blood in EDTA tube and Clotted blood tubes?

A

Different analytes after centrifuge

EDTA = plasma, buffy coat (WBC) and red cells (intact)
» For plasma DNA (extracellular DNA) e.g. prenatal screening for Down syndrome

Clotted blood tube = Clotted cells, serum
» For serum protein for ELISA

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9
Q

BRCA mutation testing.

2 methods of sample collection?

A

Buccal cell by buccal swab or saliva collector

Whole blood

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10
Q

DNA and RNA can be extracted from paraffin-embedded tissue sections. True or False?

A

False. Microdissection samples RNA is decayed.

Extract high molecular weight DNA and RNA from fresh frozen tissues
Extract only DNA from paraffin-embedded tissue section s

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11
Q

Effect of genetic mutation upstream or downstream of transcription promoter?

A

Upstream of promoter = Change level of expression

Downstream of promoter = Abnormal nucleotide sequence, abnormal protein structure and function

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12
Q

6 steps in genomic DNA extraction

A

1) Cell/ Tissue lysis
2) Protein precipitation with phenol chloroform(separate
3) RNA digestion with ribonuclease
4) DNA precipitation with absolute alcohol
5) Wash with 70% alcohol
6) DNA renaturation with Tris-EDTA buffer

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13
Q

3 cautions during genomic DNA extraction

A
  • avoid rapid stirring to damage DNA by shear force
  • Autoclave glassware and solutions to destroy Deoxyribonuclease (DNase) activity
  • Dissolve DNA in buffer with EDTA to chelate Mg ions needed for DNase activity
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14
Q

6 steps in genomic RNA extraction

A

1) Cell/ tissue lysis
2) Inactivate endogenous RNase activity with inhibitors (guianidine thiocyanate and B-mercaptomethanol) (cf inactivate RNA activity with ribonuclease in DNA extraction)
3) Protein precipitation with phenol chloroform
4) RNA precipitation with isopropanol/ lithium chloride (cf abolsulte alcohol in DNA extraction)
5) Wash with 70% alcohol
6) Resuspend RNA in DEPC- treated water (cf suspend DNA in EDTA buffer)

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15
Q

2 cautions during genomic RNA extraction

A
  • RNA is easily digested by ribonucleases (RNase)

- RNase are difficult to be inactivated, heat-stable

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16
Q

Name one example of a kit used for DNA extraction.

Advantage over conventional method?

A

QIAamp DNA blood kit

  • Simplified procedure, no phenol chloroform needed for protein precipitation
  • DNA binds to silica membrane > pure DNA eluted in buffer or water
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17
Q

Name one example of a kit used for RNA extraction.

Advantage over conventional method?

A

QIAamp RNA blood mini kit

  • Simplified procedure, no phenol chloroform needed for protein precipitation
  • Nucleic acid binds to silica membrane
  • Pure RNA eluted in water
18
Q

Assessment of high quality genomic DNA and RNA after gel electrophoresis and staining.

A

High quality DNA: intact smear band

High quality RNA: presence of 18s and 28s ribosomal RNA

19
Q

Principle of gel electrophoresis

A

Use voltage to drive movement of charged molecules > separate DNA and RNA molecules by size

20
Q

2 materials for gel electrophoresis

A

Agarose, Polyacrylamide

21
Q

agarose gel for electrophoresis

  • Adv
  • Disadv
  • Composition
  • Use
  • concentration
A

Adv:

  • Cheap, non-toxic
  • Powder form

Disadv:
- Fragile

Highly purified polysaccharide derived from agar

Separating nucleic acid

Concentration: 0.4% - 4%, determines pore size. Higher % = higher resolution

22
Q

Polyacrylamide gel for electrophoresis

  • Adv
  • Structure
A

Adv: physically tougher than agarose gel

Structure: Acrylamide monomer co-polymerizes with cross-linker Bisacrylamide

via TEMED catalyst and Ammonium persulphate (start polymerization)

23
Q

Function of electrophoresis buffer and voltage change

Examples of gel electrophoresis buffer

A

Voltage: changes rate of charged molecule movement

Buffer:

  • Stabilizes pH of matrix
  • Optimal ionic strength/ concentration to control current and heat production (not melt gel)
  • Never bind with DNA and RNA

Acetate, citrate, phosphate, Tris, EDTA

24
Q

High concentration buffer was used to run gel electrophoresis.
Problems?
Fix?

A

Problems:

  • Melt gel
  • Uneven distribution of heat distorts band shapes

Solutions:

  • Run at lower voltages
  • Run in refrigerated buffers
25
Q

Factors that determine sample migration in gel electrophoresis ?

A

Voltage
Buffer pH, concentration, ionic strength

Sample:

  • Charge - high net charge = faster migration
  • Size - smaller size = less frictional and electrostatic force = faster migration
  • Shape - affect frictional and electrostatic force
26
Q

2 solutions added to DNA/ RNA samples before loading into gel?

2 solutions added after gel electrophoresis?

A 
Sucrose or Glycerol to increase sample density - sink to bottom of gel well 
Marker dye (e.g. bromophenol blue) for observation

Ethidium bromide - bind to DNA and fluoresces with orange color under UV
SYBR safe DNA gel stain

27
Q

PCR reaction mix content

3 phases of PCR reaction

A 
Target DNA as template 
Forward and reverse primer 
dNTP 
Reaction buffer 
Magnesium ion (cofactor of DNA polymerase)
DNA polymerase
  1. Denaturation (94 degrees) - DNA strands separate
  2. Annealing (50-60) - Primer bind to template
  3. Extension (72) - Primers extended
28
Q

4 steps in Sanger sequencing

A
  1. DNA sample divided into 4 separate sequencing reactions > only 1 of 4 dideoxynucleotides is added to each reaction
  2. Initiation of strand synthesis
  3. Strand synthesis terminates with dideoxynucleotide is added
  4. Electrophoresis and autoradiography
29
Q

Automated DNA sequencing principle

A

Each ddNTP is labeled with different fluorescent probe
ddA, ddT, ddC, ddG have different colors

Sequence is represented by peaks and color

30
Q

Reverse transcription

  • Principle
  • Applications (4)
A
  • Generate cDNA from RNA&raquo_space; serve as DNA template for PCR

Applications:
- PCR analysis - detect deletion in causative gene
- Restriction fragment length polymorphism (RFLP) - detect genomic variations
- RT-PCR analysis - detect mutation at splice junctions causing intron inclusion
- RT-PCR analysis - compare gene expression between tumour and non-tumour tissue

31
Q

Describe Restriction fragment length polymorphism (RFLP)

A

PCR + restriction enzyme digestion

If normal band size is 100bp
Mutation occurs on one end of band > band size increases after digestion
Mutation occurs between two ends > create two short bands after digestion

32
Q

FISH

  • Principal
  • Function
A

Detect and localize presence or absence of specific gene sequence on chromosome by fluorescent probes complimentary to target DNA sequence

Visualize chromosomal abnormalities (e.g. translocation, deletion, amplification)

33
Q

FISH procedure

A
  1. Denature chromosome
  2. Denature probe (with complimentary sequence to target DNA)
  3. Hybridization to cell nucleus
  4. Fluorescence staining
  5. Examine slides
34
Q

Interpretation of FISH fluorescent dots?

A
  • Duplication in the region of interest > gain of fluorescent dots (e.g. HER2 amplification in breast cancer = clusters of dots in cancer cells)
  • Diploid dots for fusion (e.g. BCR/ ABL1 gene fusion in CML)
  • Lack of dots for deletion
35
Q

Advantages of FISH

A
  • Can be used in formalin-fixed, paraffin embedded sections or fresh frozen tissue
  • Simultaneous detection of multiple gene sequences
  • Less labor-intensive way to show DNA segment within entire genome
36
Q

Karyotyping

  • Principal
  • Function
A

Chart of chromosome pairs in metaphase

Study the number and appearance of chromosomes in nucleus
Find abnormalities e.g. trisomy 21

37
Q

Karyotyping Procedure

A
  1. Sample cell in growth medium (+ mitogen)
  2. Mitotic inhibitor
  3. Hypotonic solution and centrifuge separation
  4. Fix samples onto slides
  5. Banding: identify chromosomes by specific banding patterns
  6. Karyotyping: arrange chromosomes according to length and location of centromere
38
Q

Alternative to conventional karyotyping

A

Fluorescence karyotyping

  • Specific probes to chromosomes with different colours
  • Sorting done by computer
39
Q

ELISA
- Principal

  • Advantage
A

Enzyme- linked Immunosorbent Assay

Detect and quantify proteins, Antibodies, peptides or hormones in sample

Handles large quantity of samples

40
Q

ELISA

- 3 types

A
  1. Direct ELISA
    Primary antibody attach to plate via silver ion, binds to analyte directly
  2. Indirect ELISA
    Primary antibody bind to plate via silver ion. Secondary antibody bind to analyte
  3. Sandwich ELISA
    Capture antibody coated to plate. Target substrate sandwiched between secondary antibody conjugate and capture antibody

MBBS IV - Supplementary Courses (54 decks)

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