V. Lab | 78. Genotyping of the TAS2R38 tasting receptor by PCR-RFLP Flashcards
I. Background information
1. What are the features of the TAS2R38 gene?
The TAS2R38 gene has 3 SNPs = 3 nucleotides affected by polymorphisms (missense mutation):
- Dominant ‘’T’’ allele codes for the PAV allelic variant containing Proline-Alanine- Valine amino acids in the variable positions -> this receptor is sensitive to PTC
- The recessive ‘’t’’ allele encodes the AVI variant containing Alanine-Valine- Isoleucinethis receptor is PTC insensitive
I. Background information
2. How many genotype variations possible determining the phenotype?
3 genotype variations are possible in determining the phenotype:
- TT homozygotes (2x PAV) = taster
- Tt heterozygotes (PAV + AVI) = taster
- tt homozygotes (2x AVI) = non-taster
I. Background information
3. What are the 4 steps of Genotyping of the TAS2R38 bitter taste receptor by PCR-RFLP?
- Isolation of genomic DNA from buccal cells
- Setting up PCR reactions – DNA amplification
- Restriction digestion (RFLP = restriction fragment length polymorphism)
- Agarose gel electrophoresis
II. Step 1 - Isolation of genomic DNA from buccal cells
1. What are the steps in isolation of genomic DNA from buccal cells?
- genomic DNA will be prepared by abrasing the inner surface of cheeks/outer surface of gums using a cotton swab
- cut off the cotton bud of the buccal swab -> place it into an Eppendorf tube and add PBS (phosphate buffered saline)
- add proteinase K -> destroy the enzymes that would destroy the DNA (exo- + endonucleases)
- add genomic lysis/binding buffer -> helps us get rid of the cell membrane
- vortex and incubation at 55 degrees (temperature optimum for proteinase K)
=> Genomic DNA will be isolated from cell lysates using affinity chromatography.
II. Step 1 - Isolation of genomic DNA from buccal cells
2. Where do we place the cotton bud after cutting off?
cut off the cotton bud of the buccal swab
=> place it into an Eppendorf tube and add PBS (phosphate buffered saline)
II. Step 1 - Isolation of genomic DNA from buccal cells
3. What is the purpose of adding proteinase K?
destroy the enzymes that would destroy the DNA (exo- + endonucleases)
II. Step 1 - Isolation of genomic DNA from buccal cells
4. What is the purpose of adding genomic lysis/binding buffer?
helps us get rid of the cell membrane
II. Step 1 - Isolation of genomic DNA from buccal cells
5. At what temperature that we need to incubate the mixture at? Why?
vortex and incubation at 55 degrees (temperature optimum for proteinase K)
II. Step 1 - Isolation of genomic DNA from buccal cells
6. Which technique we need to use in order to isolate genomic DNA from cell lysates?
Genomic DNA will be isolated from cell lysates using affinity chromatography.
II. Step 1 - Isolation of genomic DNA from buccal cells
7. What is the principle of DNA affinity chromatography?
- The principle of DNA affinity chromatography is that a cationic matrix enclosed within a plastic cylinder binds negatively charged nucleic acids with high affinity, while other components of cell lysates do not/weakly attach to the surface of the matrix.
- 3 phases:
1) absorption (binding): DNA binds to gel surface
2) wash: wash away weakly bound impurities using appropriate buffers
3) elution: collection of DNA with alkalic buffer
II. Step 1 - Isolation of genomic DNA from buccal cells
8. What are the 3 phases of affinity chromatography?
1) absorption (binding): DNA binds to gel surface
2) wash: wash away weakly bound impurities using appropriate buffers
3) elution: collection of DNA with alkalic buffer
(The principle of DNA affinity chromatography is that a cationic matrix enclosed within a plastic cylinder binds negatively charged nucleic acids with high affinity, while other components of cell lysates do not/weakly attach to the surface of the matrix. )
III. Step 2 - Setting up PCR reactions – DNA amplification
1. What are the 3 phases of a thermocycle of a polymerase chain reaction (PCR)?
- Denaturation: dsDNA will be heated and denatured at 95 degrees to yield single-stranded templates
- Annealing: sequence specific ssDNA primers are hybridized at lower temperature (55 degrees) to the tempate DNA, marking the border of the region to be amplified
- Extension: elongation of primers occur at 72 degrees -> where DNA polymerase works the best
=> initial denaturation: 95 - 98 degrees
III. Step 2 - Setting up PCR reactions – DNA amplification
2. How many time does this thermocycle need to be repeated?
This thermocycle is repeated 40 times.
=> The amount of PCR products will be doubled in each
cycle, so about 40 cycles
=> 240 = need a lot of DNA for the electrophoresis
III. Step 2 - Setting up PCR reactions – DNA amplification
3. How does DNA polymerase operate in PCR reactions?
DNA polymerases are unable to start the synthesis of the complementary (coding) strand,
therefore, a primer with a free 3’-OH end is required so the elongation can be catalyzed
III. Step 2 - Setting up PCR reactions – DNA amplification
4. What does the PCR mixture consist of?
- DNA template
- DNA polymerase
- 2x primers (forward and reverse)
- Nucleotides
- Buffer
IV. Step 3 - Restriction digestion
(RFLP = restriction fragment length polymorphism)
1. Why do we need to perform restriction digestion?
- Perform restriction digestion to differentiate between the 2 alleles.
- The restriction endonuclease will cleave one of the alleles in a sequence-specific manner, but does not recognize the other sequences that contain a single base variation.
- The PCR product of one of the allele variants will be cleaved to yield 2 shorter fragments, transforming the sequence variation into length difference that can be visualized by gel electrophoresis.
IV. Step 3 - Restriction digestion (RFLP = restriction fragment length polymorphism)
2. What is the mechanism of Restriction digestion?
- The restriction endonucleases recognize 4-8 bp long palindromic sequences in the DNA double helix and cleave both strands by hydrolysis of phosphodiester bonds.
- Sticky ends = different length cleavage, blunt ends = same length cleavage.
IV. Step 3 - Restriction digestion (RFLP = restriction fragment length polymorphism)
3. What are the purpose and mechanism of Fnu4HI restriction endonuclease ?
- The Fnu4HI restriction endonuclease will be used for TAS2R38 genotyping:
=> The PTC-sensing allele contains a C-nucleotide
=> The non-taster phenotype corresponds to a T genotype that cannot be cleaved by this endonuclease
=> Restriction digestion performed at 37 degrees for an hour
V. Step 4 - Agarose gel electrophoresis
1. What is Agarose gel electrophoresis?
DNA fragments will be separated and visualized by agarose gel electrophoresis
V. Step 4 - Agarose gel electrophoresis
2. What are the steps of Agarose gel electrophoresis?
- DNA fragments will be separated and visualized by agarose gel electrophoresis
- Separation is based on size only => the smallest fragments travel the furthest (to the positive pole) and the largest the shortest (close to negative pole)
- Loading buffer (consisting of glycerol, bromophenol blue, xylenone) is added to the
digestion product - To determine the approximate size (molar mass) of digestion products, a DNA ladder (molar mass marker) will also run in a separate lane of the gel => DNA marker contains 10 DNA fragments of different molar masses
- Running process takes about 30 minutes at 120 V
- Gel slabs are trans-illuminated by means of a UV lamp following the electrophoretic separation
V. Step 4 - Agarose gel electrophoresis
2. What is Agarose?
Agarose is a polysaccharide isolated from marine algae (seaweed) whose chains
readily undergo hydration, forming an amorphous gel with numerous pores enabling
it to function as a molecular sieve
V. Step 4 - Agarose gel electrophoresis
3. What is separation based on?
Separation is based on size only
=> the smallest fragments travel the furthest (to the positive pole) and the largest the shortest (close to negative pole)
V. Step 4 - Agarose gel electrophoresis
4. What does the loading buffer contain?
consisting of glycerol, bromophenol blue, xylenone
V. Step 4 - Agarose gel electrophoresis
5. How do we determine the approximate size (molar mass) of digestion products?
To determine the approximate size (molar mass) of digestion products, a DNA ladder (molar mass marker) will also run in a separate lane of the gel
=> DNA marker contains 10 DNA fragments of different molar masses
V. Step 4 - Agarose gel electrophoresis
6. What is DNA marker?
molar mass marker
V. Step 4 - Agarose gel electrophoresis
7. What can we conclude from Agarose gel electrophoresis?
- In the case of the TT homozygous genotype (non-taster phenotype), the amplicon cannot be cleaved by the endonuclease. Uncut 303 bp band can be seen at the same height as the 300 bp marker band
- CC homozygotes (taster phenotype) produce 2 restriction fragments with 239 and 64 bp molar masses
- In the case of CT heterozygotes (taster phenotype due to presence of the dominant allele), both the 303 bp and the 239 bp fragments can be seen
V. Step 4 - Agarose gel electrophoresis
8. In the case of the TT homozygous genotype
=> What can we observe?
- In the case of the TT homozygous genotype (non-taster phenotype), the amplicon cannot be cleaved by the endonuclease.
- Uncut 303 bp band can be seen at the same height as the 300 bp marker band
V. Step 4 - Agarose gel electrophoresis
9. In the case of the CC homozygotes
=> What can we observe?
CC homozygotes (taster phenotype) produce 2 restriction fragments with 239 and 64 bp molar masses
V. Step 4 - Agarose gel electrophoresis
10. In the case of the CT heterozygotes
=> What can we observe?
In the case of CT heterozygotes (taster phenotype due to presence of the dominant allele), both the 303 bp and the 239 bp fragments can be seen
