Genotyping of ALDH2 gene by allele-specific PCR

Aldehyde dehydrogenase

• reaction

  • CH3CH2OH → (by alcohol dehydrogenase) CH3CHO (acetaldehyde) → (by ALDH2) CH3COO- (acetate)

• deficiency of ALDH2

  • acetaldehyde cannot be converted to acetate

  • → accumulation of acetaldehyde

  • → alcohol flush response

• SNP in ALDH2 gene

  • rs671: SNP (G to A) at codon 504

  • E504K: amino acid translated changes from Glu to Lys

Allele-specific PCR

General SNP genotyping

• to determine the SNP allele at each SNP marker in each sample

• methods includes

  • allele-specific PCR

  • PCR-RFLP

  • DNA sequencing

  • microarray

  • primer extension

PCR——polymerase chain reaction

• purpose: amplify a specific DNA fragment from a complex mixture of DNA molecule

• procedures

  • strand separation (95°C)

    • heat denaturation of template DNA

  • primer annealing (58°C)

    • forward primer: same as the top strand

    • reverse primer: reverse complement of the top strand

  • strand elongation (72°C)

    • DNA synthesis always proceeds from 5’ to 3’ end of the growing strand

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

    • Mg2+ is required as cofactor

• ★ PCR products contain primer sequences

General info of Allele-specific PCR

• also known as amplification refractory mutation system (ARMS)

• use allele-specific primer for PCR

  • presence or absence of PCR product indicate the presence or absence of the target allele

  • mismatch at 3’-OH terminal of primer→ drastically reduce amplification efficiency to differentiate different alleles

• require DNA polymerase without 3’ to 5’ exonuclease activity

  • proofreading activity of DNA polymerase remove the mismatched base at the 3’ end of the growing DNA → cannot differentiate the alleles

• Components require for the reaction

  • buffer

  • template DNA

  • primers

  • dNTPs

  • thermostable DNA polymerase

  • divalent cation: Mg2+ for catalytic activity of DNA polymerase

    • may cause min incorporation of bases if Mn2+ is used

Primer design

• avoid formation of primer dimers: no complementary sequences on 3’ ends of both primers

  • primer dimers= primers anneal to each other

  • this leads to fewer primers available for annealing

• avoid inverted repeat in primers

  • → primer self-annealing and no primer extension

• 18-30 nucleotides long

• PCR annealing temperature

  • melting temperature of primer= (no. of A + T)*2°C + (no. of G + C)*4°C [for short oligonucleotides up to 20 bases]

  • melting temperature of forward and reverse primer should not differ by > 5°C

  • set the temperature of annealing a few degrees below the melting temperature

    • too high: primer may not anneal → poor amplification

    • too low: tolerate mismatch between primer and template → non-specific amplification

• additional deliberate mismatches at the penultimate base of the primer

  • increase discrimination between alleles

Multiplex PCR

• amplify more than one gene in a single PCR reaction

• serving as positive control: to show the PCR is functioning → the absence of PCR product in allele-specific PCR is not due to

  • failure of PCR or

  • absence of genomic DNA template

Agarose gel electrophoresis

Principle

• for analysis of DNA and purification of DNA fragments for cloning

• usually run submerged in running buffer

• DNA or RNA are -ve charged: migrate towards +ve electrode when in electric field

• DNA or RNA molecules have a constant charge-to-mass ratio

  • migrate according to their size

  • smaller molecules migrate faster

  • mobility of DNA is inversely proportional to the log of the number of pairs in DNA

Procedures

• add DNA samples and DNA ladder (size marker) onto agarose gel lane

• DNA bands are separated by size from negative electrode to positive electrode

• add dye to bind to DNA

• expose DNA bands on film: bands become visible Unser UV light

Equipment required

• agarose

  • a liner polysaccharide made up of the basic repair unit agarobiose

  • solution in hot water: gel is formed via the corsslinking of agarose polymer chain by interchain hydrogen bond when cools down

  • size of pores: 50-200 nm (depends on concentration of agarose)

    • As the agarose concentration increases, the average diameter of the pore decreases

  • optimal resolution of DNA depends on concentration of agarose

• electrophoresis chamber and power supply

• gel casting tray

• sample combs

  • around which molten medium is poured to form wells in gel

• electrophoresis buffer

  • TAE

    • lower buffering capacity

    • may need recirculation of buffer

    • preferred if extraction of DNA from gel is needed or separating large DNA molecules

  • TBE

    • higher buffering capacity

    • used DNA recovery is not needed

    • slower DNA migration but sharper bands are produced

  • pH 8.3

  • usually prepared as 10X or 5X and dilute to 1X working concentration when use

  • must use the same buffer for gel preparation and electrophoresis

• loading dye or buffer

  • contains glycerol, Ficoll or sucrose to provide sample density for loading

  • 6X or 10X concentrated form

  • contains tracking dyes to monitor the progress of electrophoresis

    • xylene cyanol co-migrates with DNA fragments ~ 4kb (slowest)

    • bromophonel blue co-migrates with DNA fragments ~ 0.4 kb

    • orange G co-migrates with DNA fragments ~ 50bp (fastest)

• DNA intercalating dye

  • include in gel or/and buffer to visualise DNA

  • stain gel with DNA binding dye after electrophoresis to visualise DNA

  • higher affinity to double stranded DNA

  • Radiation at 302 nm and 366 nm is absorbed by dye → re-emits as fluorescence at 590 nm (red-orange)

  • examples

    • ethidium bromide

    • GelRed

    • other dyes such as SYBR Gold, SYBR Safe, SYBR Green and methylene blue

• UV transilluminator

  • visualise DNA at 302 or 366 nm

  • photography with gel documentation system

  • determine the approx. length of DNA molecule by comparing their migration to that of standards

Factors affecting agarose gel electrophoresis of DNA

• DNA size (inversely proportional to the lot of the number of base pair)

• concentration of agarose

  • different concentration for different size range

• voltage

  • Migration of DNA on agarose gel roughly proportional to voltage x hour

  • use lower voltage for running larger size of DNA fragments

• DNA topological form

  • nicked circular: slower

  • linear (size marker is designed for linear DNA fragments only)

  • superhelical: faster