Molecular Genetics - Techniques for genetic analysis (I)

Question 1

Define molecular pathology

Answer 1

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

Question 2

Application of molecular pathology in Oncology

Answer 2

• 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

Question 3

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

Answer 3

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

Question 4

Give one example of molecular pathology test for viruses and application

Answer 4

HPV

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

Application: Screen for cervical cancer, monitor disease recurrence

Question 5

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

Answer 5

BRCA 1/2 mutation screening for breast or ovarian cancer

Question 6

Give one example of molecular pathology test for toxicology screening

Answer 6

Predict adverse drug reaction

e.g. Carbemazepine need HLA typing

Question 7

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

Answer 7

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

Question 8

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

Answer 8

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

Question 9

BRCA mutation testing.

2 methods of sample collection?

Answer 9

Buccal cell by buccal swab or saliva collector

Whole blood

Question 10

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

Answer 10

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

Question 11

Effect of genetic mutation upstream or downstream of transcription promoter?

Answer 11

Upstream of promoter = Change level of expression

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

Question 12

6 steps in genomic DNA extraction

Answer 12

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

Question 13

3 cautions during genomic DNA extraction

Answer 13

• 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

Question 14

6 steps in genomic RNA extraction

Answer 14

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)

Question 15

2 cautions during genomic RNA extraction

Answer 15

• RNA is easily digested by ribonucleases (RNase)

- RNase are difficult to be inactivated, heat-stable

Question 16

Name one example of a kit used for DNA extraction.

Advantage over conventional method?

Answer 16

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

Question 17

Name one example of a kit used for RNA extraction.

Advantage over conventional method?

Answer 17

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

Question 18

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

Answer 18

High quality DNA: intact smear band

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

Question 19

Principle of gel electrophoresis

Answer 19

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

Question 20

2 materials for gel electrophoresis

Answer 20

Agarose, Polyacrylamide

Question 21

agarose gel for electrophoresis

• Adv
• Disadv
• Composition
• Use
• concentration

Answer 21

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

Question 22

Polyacrylamide gel for electrophoresis

• Adv
• Structure

Answer 22

Adv: physically tougher than agarose gel

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

via TEMED catalyst and Ammonium persulphate (start polymerization)

Question 23

Function of electrophoresis buffer and voltage change

Examples of gel electrophoresis buffer

Answer 23

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

Question 24

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

Answer 24

Problems:

• Melt gel
• Uneven distribution of heat distorts band shapes

Solutions:

• Run at lower voltages
• Run in refrigerated buffers

Question 25

Factors that determine sample migration in gel electrophoresis ?

Answer 25

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

Question 26

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

Answer 26

``` 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

Question 27

PCR reaction mix content 3 phases of PCR reaction

Answer 27

``` 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

Question 28

4 steps in Sanger sequencing

Answer 28

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

Question 29

Automated DNA sequencing principle

Answer 29

Each ddNTP is labeled with different fluorescent probe ddA, ddT, ddC, ddG have different colors Sequence is represented by peaks and color

Question 30

Reverse transcription - Principle - Applications (4)

Answer 30

- Generate cDNA from RNA >> 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

Question 31

Describe Restriction fragment length polymorphism (RFLP)

Answer 31

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

Question 32

FISH - Principal - Function

Answer 32

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)

Question 33

FISH procedure

Answer 33

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

Question 34

Interpretation of FISH fluorescent dots?

Answer 34

- 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

Question 35

Advantages of FISH

Answer 35

- 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

Question 36

Karyotyping - Principal - Function

Answer 36

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

Question 37

Karyotyping Procedure

Answer 37

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

Question 38

Alternative to conventional karyotyping

Answer 38

Fluorescence karyotyping - Specific probes to chromosomes with different colours - Sorting done by computer

Question 39

ELISA - Principal - Advantage

Answer 39

Enzyme- linked Immunosorbent Assay Detect and quantify proteins, Antibodies, peptides or hormones in sample Handles large quantity of samples

Question 40

ELISA | - 3 types

Answer 40

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