Genotyping of TAS2R38 (PTC) gene by PCR-RFLP and DNA sequencing

TAS2R38——PTC

Characteristics

• bitter taste of PTC is mediated bt the T2T38 receptor on taste bud

  • PAV-type: able to taste bitter

  • AVI-type: X taste bitter

• ability to taste PTC (phenylthiocarbamide) is a polymorphic trait mediated by TAS2R38 (PTC) bitter taste receptor gene

  • able to taste PTC: dominant trait

Polymorphism of TAS2R38

• 3 common SNPs in TAS2R38 gene

  • rs713598

    • C145G

    • Gly49→Ala

  • rs1726866

    • C785T

    • Ala262Val

  • rs10246939

    • G886A

    • Val296Ilu

• haplotype: a specific combination of neighbouring alleles from different polymorphisms that tend to be inherited tgt

  • functional haplotype

    • PAV

    • AAV

    • PVI

    • AAI

  • non-functional haplotype

    • AVI

PCR-RFLP

Principle

• a PCR-based method to detect nucleotide polymorphism

• also known as cleaved amplified polymorphic sequence assay

Procedures

• PCR primers are used to amplify a DNA fragment containing the polymorphic sites

• PCR products are digested with restriction enzyme (Fnu4HI)

  • differentiate the allele by cutting the target sites if present

  • → restriction fragment length polymorphism

• analysis by gel electrophoresis

DNA purification

Purpose

• purify DNA from cells or agarose gel pieces

• remove unincorpotrated dNTPs, primers, salt from PCR reaction

Principle of DNA purification by adsorption to silica surface

• based on the selective adsorption of DNA to silica in the presence of a high concentration of chaotropic salt (guanidine HCl)

  • → DNA stick to silica by forming a cation bridge

• can be eluted using water or low-salt buffer

QIAquick PCR purification kit

• buffer PB

  • guanidine HCl in 30% isopropanol to promote binding of DNA to silica matrix

  • ensure colour of mixture is yellow: pH≤ 7.5 (adjust with sodium acetate if necessary)

    • DNA binding to silica surface is pH dependent: inherently greater affinity for the surface in acidic environment

• buffer PE

  • contain 80% ethanol to wash column to remove weakly bound contaminants

• buffer EB

  • a low salt buffer to elute DNA

  • should be added directly onto the membrane

Other common methods for DNA purification

• anion-exchange chromatography

  • -ve phosphates on DNA interact with +ve on surface of resins

  • RNA proteins, carbohydrates and other metabolites bound less tightly to the resin → washed away by medium-salt buffer

  • DNA is eluted with high-salt buffer

• precipitation with salt and ethanol

  • salt and ethanol remove water around DNA

  • salt neutralises the charge on DNA with the presence of ethanol → DNA becomes less hydrophilic

  • ethanol promote aggregation of DNA

  • DNA precipitate and can be obtained by discarding the supernatant

• organic solvent extraction using phenol or chloroform

  • Mix the DNA sample with the organic solvent.

  • Centrifuge the mixture to separate the aqueous and organic phases.

  • Biomolecules partition between the two phases based on their affinity for the solvent

  • transfer and retain aqueous phase DNA

• ultracentrifugation——equilibrium density-gradient centrifugation

  • under centrifugal force: particles move until their density is the same as the surrounding medium

DNA sequencing

Sanger method

• also known as chain termination method

• based on DNA synthesis

  • require a primer strain a template strand and dNTPs

• DNA polymerase adds dNTPs to the free 3’-OH end of primer strand

• incorporation of dideoxyribonucleotide (ddNTP) → termination of chain elongation due to no available 3’-OH

  • as concentrations of ddNTPs were adjusted: chain termination occurs randomly at one of many complementary positions

  • different fluorescent dyes for each ddNTP

• sequencing products are separated by electrophoresis on a slab gel or capillary

  • detect different colour of fluorescent labels by laser excitation

  • → identify the terminal bases

Cycle sequencing

• used the technique of sanger sequencing

Reaction mixture

• DNA polymerase

• dNTPs

• buffer

• dye-labeled terminator

• primer

• template strand

Procedures

• annealing (50-55°C)

  • a sequencing primer anneals to the DNA template strand

• extension (60-70°C)

  • DNA polymerase extends the primer by adding dNTPs complementary to the template strand

  • a trace amount of ddNTPs with different fluorescent dye is added as well

    • incorporation leads to chain termination

• cycle repeats for amplification

• electrophoresis to separate extension products

• identification o0f terminator by dye-specific fluorescence emission