Single nucleotide polymorphism (SNP) detection by polymerase chain

reaction (PCR)-restriction fragment length polymorphism (RFLP)

Masao Ota1*, Hirofumi Fukushima1, Jerzy K. Kulski2,3, Hidetoshi Inoko3

1) Department of Legal Medicine, Shinshu University School of Medicine, Nagano,

390-8621, Japan.

2) Centre for Comparative Genomics, School of Information Technology, Murdoch

University, Murdoch, Western Australia 6150, Australia

3) Department of Molecular Life Science, Division of Molecular Medical Science

and Molecular Medicine, Tokai University School of Medicine, Kanagawa,

259-1143, Japan

* Corresponding Author: Masao Ota: e-mail; otamasao@sch.md.shinshu-u.ac.jp,

Tel:81-263-37-3217

ABSTRACT

The accurate analysis of DNA sequence variation in not only humans and animals but

also other organisms has played a significant role in expanding our knowledge about

genetic variety and diversity in a number of different biological areas. The search for an

understanding of the causes of genetic variants and mutations has resulted in the

development of a simple laboratory technique, known as the polymerase chain

reaction-restriction fragment length polymorphism (PCR-RFLP) method, for the

detection of single nucleotide polymorphisms (SNPs). PCR-RFLP allows rapid

detection of point mutations after the genomic sequences are amplified by PCR. The

mutation is discriminated by digestion with specific restriction endonucleases and is

identified by gel electrophoresis after staining with ethidium bromide. This convenient

and simple method is inexpensive and accurate for SNP genotyping and especially

useful in small basic research studies of complex genetic diseases. The whole protocol

takes only a day to carry out.

Introduction

The application and refinement of the PCR method since its invention in the 1980s1-3

has dramatically progressed molecular genetic research. This simple and sensitive

enzymatic technique for the amplification of DNA fragments has been used for various

purposes, but especially for the detection of nucleic acid polymorphisms to find

biological meaning in genetic variation and molecular divergence in living organisms.

The majority of polymorphisms in the human genome are single nucleotide

polymorphisms (SNPs) that account for more than 90% of sequence variation4 and are

utilized as an important tool for the study of the structure and history of our genome.

There are more than 27 million SNPs in the human genome that have been recorded and

stored in the SNPs database (http://www.ncbi.nlm.nih.gov/SNP). This immense amount

of data provides valuable information for SNP-based studies5, identification of

candidate genes involved with complex genetic diseases6-8, pharmacogenetic analysis9-10,

drug development11, population genetics12-13, evolutionary studies14 and forensic

investigations15.

Over the past twenty years, many different methods have been developed for SNP

genotyping by PCR, including hybridization16, allele-specific PCR17, primer extension18,

oligonucleotide ligation19, direct DNA sequencing20 and endonuclease cleavage after

amplification of the subjected genomic region by PCR21-22. Recent technologies for

SNP genotyping (Taq Man method23-24, Invader method25-26, MALDI-TOF method27,

GeneChips28) have the potential for high-throughput, but they require the purchase of

expensive equipment. Often, however, the throughput needs for SNP detection may be

relatively low as this will depend mostly on the aim of the study.

One inexpensive, simple and convenient method for SNP genotyping is PCR-RFLP and

its utility in molecular genetic studies has been demonstrated in a wide range of fields.

Since 1988 (see ref. 21), more than 3700 reports related to PCR-RFLP have been cited

in the PubMed bibliographic information database at NCBI

(http://www.ncbi.nlm.nih.gov/entrez/query.fcgi).

PCR-RFLP is based on endonuclease digestion of PCR-amplified DNA. The

specific restriction endonuclease recognizes and cleaves the DNA in the region of the

point mutation of the PCR products. The SNP type is easily identified by using gel

electrophoresis to confirm and separate the sizes of the smaller DNA fragments

generated by the endonuclease digestion. The PCR-RFLP is a simple, sensitive and

reliable method and requires minimal investment in instrumentation. The limitations of

this protocol are in the cases such that the sequences of target SNPs are not suitable for

commercial restriction enzymes, and inversely, that the sequences have too many

recognition sites for a single restriction enzyme. This method is available for

genotyping not only a SNP, but also insertion/deletion polymorphisms29-31 and multiple

mutations such as Human Leukocyte Antigen (HLA) alleles22,32-33 (Figure 1), which

consist of multiple assemblies of SNPs. The ABO blood group polymorphism was the

first molecular polymorphism to have been observed and analyzed in humans. This
antigenic variation for red blood cells is due to a few DNA single-base substitutions that

produce the A, B, and O alleles that can be identified as the ABO genotypes by the

PCR-RFLP method34 (Figure 2).

Experimental Design

Choice of Restriction Enzymes and design of PCR Primers for PCR-RFLP.

PCR-RFLP always starts with the need to design an optimum primer pair and to find the

restriction enzymes that will identify the SNPs in the PCR-amplified product. To obtain

high quality results, it is important to design specific amplification primers and select an

appropriate restriction enzyme from the REBASE Restriction Enzyme Database35

(http://rebase.neb.com). To assist with this potentially difficult and tedious step,

several comprehensive and automated search tools have been provided by web

interfaces that are available at http://cedar.genetics.soton.ac.uk/public_html/primer (see

ref. 36) or http://bioinfo.bsd.uchicago.edu/SNP_cutter.htm (see ref. 37). These

programs are capable of designing primers for either natural PCR-RFLP or mismatch

PCR-RFLP, depending on the sequence data of the targeted genomic region; natural

PCR-RFLP can be used if discriminative restriction sites are found at the SNP site,

otherwise, additional mismatched bases are introduced to adjacent sites in order to

provide artificial restriction sites (known as mismatch PCR-RFLP); even if the target

SNP has no cleavage site for available restriction enzymes, it is possible to use the

mismatch PCR-RFLP method38 (Figure 3). This method uses a primer containing an

artificial restriction site (an additional mismatched base) adjacent to the SNP site. PCR

primer sets should be designed to amplify a given DNA region but not to amplify an

orthologous non-specific region or a paralogous (duplicated) region. Of course, it is

alternatively possible to manually find an appropriate restriction enzyme and to design

the corresponding primers using either commercial software (e.g. Genetyx, Genetyx Co,

Japan; DNASIS Pro, Hitachi Software Engineering Co. Ltd., Japan; Gene Construction

Kit, Textco BioSoftware, Inc., USA; DNADynamo, Blue Tractor Software, Ltd., UK) or

free software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi (see ref. 39),

http://www2.eur.nl/fgg/kgen/primer/SNP_Primers.html). To find the appropriate

restriction sites and amplification primers for producing PCR products, a

comprehensive software tool has been created for PCR-RFLP assay design37, 40.

Template preparation for PCR-RFLP. Template DNA isolation methods for

PCR-RFLP vary considerably in the starting material (e.g., fresh or frozen animal

tissues and cells, blood, body fluids, buccal swab, paraffin-embedded tissue,

formalin-fixed tissue, insects, yeasts or bacteria). The most commonly used method

for genomic DNA preparation is based on phenol extraction and ethanol precipitation41.

Alternatively, many DNA preparation kits are now commercially available and can be

useful to obtain high quality and quantity template DNA. The purity of template DNA is

determined by calculating the ratio of absorbance at 260 nm to absorbance at 280 nm.

An A260/A280 ratio of 1.7-1.9 means pure template DNA and is better suited for PCR

procedure.

Controls. Prepare at least one positive control for amplifying each of the alleles and one

negative control with no DNA. Amplification for a positive control and a

negative control is performed in parallel with sample in a different tube. Each

gel should also include the controls for checking enzyme function. When we

use single enzyme for single sample or multiple samples, we need to prepare
 internal control DNA which has a restriction site with each enzyme to check

the activity of each enzyme or slip of enzyme addition to a reaction tube.

Meanwhile, using multiple enzymes for single sample or multiple samples, it is

best to prepare same numbers of different internal control DNA as numbers of

enzyme. Each internal control DNA is consisted of an appropriate length PCR

products from plasmid DNA which has a restriction site by each enzyme.

MATERIALS

REAGENTS

*10xPCR buffer (usually supplied by the manufacturer with the Taq polymerase)

*Taq Polymerase (ABI: N8080160; N8880240, QIAGEN: 201203; 203203, Promega:

M7122; M5661, TaKaRa: R001A, R007A, etc.; use hot-start PCR depending on PCR

specificity)

*dNTP Mixture (dATP, dCTP, dGTP, dTTP: usually supplied by the manufacturer with

the Taq polymerase, Promega: U1511)

*PCR primers (forward and reverse: 20 µM in TE buffer; primers are usually obtained

from company (SIGMA Genosys, Invitrogen)).

*Sterile water (autoclaved)

*Template DNA (see Experimental Design for further information)

CRITICAL: 10ng to 50ng of good quality of template DNA, extracted from various

samples as described in the Experimental Design can be used for PCR-RFLP.

*Restriction Enzymes (New England Biolabs, Promega, TaKaRa)

CRITICAL: Enzyme activity drops quickly if restriction enzymes are not kept at -20 °C.

They should be kept on ice during handling and never be left on ice after use.
*Restriction Digestion Buffer (usually supplied by the manufacturer with the restriction

enzymes)

*Agarose (NuSieve 3:1 agarose) (Lonza, 50091)

CRITICAL: Usually discriminates DNA fragments less than 1,000bp. Within a given

range of differently sized DNA fragments, the smaller DNA less than 1,000 bp are best

separated in TBE buffer.

*Tris base (Sigma, T6066)

*Boric acid (Sigma, B7901)

*0.5M EDTA, pH8.0 (Sigma, E7889)

*10% (wt/vol) AP (ammonium persulfate; Sigma, A3678) prepared freshly each time.

*Acrylamide (Sigma; A3663, Wako;011-08015)

Caution: Acrylamide is highly neurotoxic and readily absorbed through the skin. Mask

and gloves should be worn when handling this chemical.

*N,N’-Methylene bisacrylamide (Sigma;M7279, Wako;138-0632)

Caution: Bisacrylamide is highly neurotoxic and readily absorbed through the skin.

Mask and gloves should be worn when handling this chemical.

*TEMED (N,N,N’,N’-tetramethylethylenediamine; Sigma; 87689) stored at 4 ºC.

*Ethidium bromide (0.5 µg/ml) (Sigma, E8751). Stock solution is prepared as 10 mg/ml

in water and stocked in light-resistant bottle at room temperature.

CAUTION: Ethidium bromide is a mutagen with potential carcinogenicity; should wear

gloves and take care in handling, storage, and disposal.

*Bromophenol Blue (BPB: Sigma, B0126)

*Xylene cyanol FF (XC: Sigma, X4126)

*Glycerol (Sigma, G5516)

*An optimal DNA size marker used in analysis for PCR products.

EQUIPMENT

*PCR tubes, 96-well multiwell plates

*PCR Thermal Cycler

*Pipettors and autoclaved tips (filter tips are better for avoiding cross-contamination)

* Horizontal minigel electrophoresis apparatus (e.g. Mupid, ADVANCE Co. Ltd.,

Tokyo, or Scie-Plas Mini Horizontal Gel Unit, Harvard Apparatus Co. Massachusetts).

*Vertical polyacrylamide gel electrophoresis apparatus (e.g. Atto Co. Tokyo, Bio-Rad

Lab. Inc. California).

*Dry block heater

REAGENT SETUP

*TE buffer, pH8.0 (Tris/EDTA): 10mM Tris-Cl, 1mM EDTA

*10 x Stock solution electrophoresis buffer (TBE), 1 liter: 108 g Tris base, 55 g Boric

acid, 40 ml 0.5M EDTA, pH8.0

CRITICAL: Store at room temperature and remake if major residues appear in solution.

*Gel-loading dye: 0.25% Bromophenol Blue (BPB: Sigma), 0.25% Xylene cyanol FF

(XC: Sigma), 50% glycerol (Sigma), 1mM EDTA (pH8.0).

*Acrylamide-bisacrylamide stock solution (29:1): prepared as an unpolymerized 30%

solution (wt/vol) in H2O as a stock solution, as follows: 29g Acrylamide, 1g

N,N’-Methylenbisacrylamide, water to 100 ml.

CRITICAL: Stock solution is protected from light and stored in the refrigerator.

Caution: Acrylamide and bisacrylamide are highly neurotoxic and readily absorbed
through the skin. Mask and gloves should be worn when handling these chemicals.

*Polyacrylamide gels: the stock acrylamide-bis solution is mixed with water and 10 x

TBE to yield the appropriate percentage, volume, and buffer concentration (1 x) (e.g. for

100 ml of 12% polyacrylamide gel; 40ml of 30 %acrylamide-bis solution + 10 ml of 10

x TBE + 49 ml of water + 1 ml of 10% AP). This solution is gently deaerated by

swirling to make a well polymerized gel. Then, 60 µl of TEMED is added. After mixing

well for a moment, pour polyacrylamide gel solution into the glass plate of the

electorphoresis apparatus.

Procedure

PCR-amplification of DNA samples • TIMING 1.5 - 2.5 h

1. PCR reactions can be set up to amplify a single DNA sample for digestion with a

single restriction enzyme (see option A), or to amplify more than one DNA sample for

digestion with multiple restriction enzymes (option B):

A. PCR set up to amplify a single DNA sample for digestion with a single

restriction enzyme.

i) To PCR-amplify a single DNA sample, set up a 20µl reaction mixture in a 0.2 ml

micro amplification tube, as follows:

component volume (µl) final concentration

10 x PCR buffer (Mg2+) 2.0 1 xPCR buffer
dNTP mix (each 2.5 mM) 1.6 0.2mM of each dNTP
Primer (forward) (100pmol/µl) 0.2 1µM forward primer
Primer (reverse) (100pmol/µl) 0.2 1µM reverse primer
Taq polymerase (5U/µl) 0.2 1 unit Taq polymerase
Sample DNA (10-50 ng/µl) 1.0 10-50 ng
DNase-free water 14.8
total 20.0

B. PCR set up to amplify more than one DNA sample for digestion with multiple
restriction enzymes.

i) To PCR-amplify more than one DNA sample, prepare a master reaction mixture in a

1.5ml tube, as follows, adding the template DNA individually to PCR tubes in advance:

component n=10* volume (µl)

+
10 x PCR buffer (Mg2 ) 22.0
dNTP mix (each 2.5 mM) 17.6
Primer (forward) (100pmol/µl) 2.2
Primer (reverse) (100pmol/µl) 2.2
Taq polymerase (5U/µl) 2.2
Sample DNA (10-50 ng/µl) 11.0
DNase-free water 162.8
total 220.0
dispense DNA sample in advance
*: add extra 10% more samples, finally make up 11 samples

CRITICAL STEP: Calculate the total volume of the master reaction mixture as the

volume required for one DNA sample multiplied by the number of DNA samples plus

an extra 10 % to account for loss of liquid during pipetting steps.

CRITICAL STEP: If the PCR products are to be digested by more than one restriction

enzyme, increase the total volume proportional to the number of enzymes that will be

used.

2. Perform PCR-amplification of template DNA using an automated thermal cycler,

with a standard amplification program, as follows, and stop PCR reactions by chilling at

4°C:

Standard PCR Hot start PCR

Initial activating step 5 min 95°C 15min 95°C
3-step cycling
Deanaturation 0.5-1 min 95°C 0.5-1 min 95°C
Annealing 0.5-1 min 55-65 °C 50-70 °C
Extension 1 min 72°C 72°C
Number of cycles 25-30 30-40
Final extension 5 min 72°C 10min 72°C
CRITICAL STEP: The program is generally designed according to manufacturer’s

instructions of Taq polymerase and primers used in PCR. Temperatures and cycling

times are optimized for individual templates and primer pairs.

Analysis of PCR Products by Gel Electrophoresis • TIMING 0.5 - 1 h

3. To separate and determine the PCR product sizes, the PCR yield and nonspecific

background, load 6 µl (5 µl of each PCR reaction product plus 1µl of gel-loading dye)

onto a 2% (w/v) agarose gel. Load an optimal DNA size marker (e.g. 100 bp ladder,

pBR322-HaeIII, φx174-Hae III, or φx174-Hinf I) in a separate lane that is parallel with

the samples on the gel.

4. Run the gel for appropriate times at constant voltage.

CRITICAL STEP: Running time depends on the expected size of the amplified product

and concentration of agarose gel. Usually electrophoresis is performed 20 min at 100 V

in 2 % agarose when the size of PCR fragment is ranging from 100 to 500 bp.

5. Stain the gel with ethidium bromide at 0.5 µg/ml for 10 minutes.

CRITICAL STEP: Alternatively, Ethidium bromide may be included in the gel and

running buffer at 0.5 µg/ml.

6. Visualize PCR products using a UV-transilluminator and record the results by

photography. Check whether the amplified product with the expected size is observed

on the gel by comparing with an optimal DNA size marker. Amplicons of the correct

size is preferable to be greater than 25 ng per band for use in the next digestion step.

(An approximate mass of PCR products is figured comparably from the intensity of the

bands with the size marker used, e.g. 100 bp ladder NEB; N3231L)

TROUBLESHOOTING
CAUTION: Safety glasses and preferably a face mask should be worn around UV light

sources.

Restriction Enzyme Digestion of PCR products • TIMING 2 - 3 h

7. The general method for digestion of PCR-amplified DNA is the addition of a

restriction enzyme directly to a reaction tube containing an aliquot of the PCR product.

Restriction enzyme digests can be set up to digest PCR products with a single enzyme

(option A) or to digest PCR products with multiple enzymes (option B):

A. Restriction enzyme digest set up to digest PCR products with a single restriction

enzyme.

i) Prepare a master-mix solution to digest a number of PCR products with a selected

restriction enzyme, as follows; each sample is reacted in a total of 10 µl of mixture

containing 5-10 units of enzyme, 1x restriction digestion buffer and 6 µl of PCR

product:

n=1 n=50*
µ µ
10 x enzyme buffer 1.0 55.0
restriction enzyme (10U /ml) 0.5 27.5
internal control DNA$ (20ng/ml) 0.5 27.5
DNase-free water 2.0 110.0
total 4.0 220.0
n: sample number
*: add extra 10% more samples, finally make up 55 samples
$: purified PCR products after amplification of plasmid DNA
which has a cleavage site with subjected restriction enzyme.

ii) Transfer 5 µl of PCR products to a PCR tube or a 96-well multiwell plate.

iii) Dispense 5µl of digestion master-mix to each well containing the PCR product.
B. Restriction enzyme digest set up to digest PCR products with multiple

restriction enzymes.

i) Prepare a master-mix solution to digest PCR products with multiple restriction

enzymes (e.g. typing HLA alleles; Figure 1), as follows:

n=1 n=10*
µ µ
10 x enzyme buffer 1.0 11.0
PCR product 6.0 66.0
DNase-free water 2.0 22.0
total 9.0 99.0
n: number of enzyme for analysis
*: add extra 10% more samples, finally make up 11 samples

CRITICAL STEP: A 10 µl volume of mastermix should be made for each restriction

enzyme and sample combination. For example, if 9 restriction enzymes are employed to

determine HLA alleles, then we would prepare a tenfold quantity of reaction mixture

based on the total volume (10 µl), for each sample

ii) Transfer 9 µl of the master-mix containing the PCR product to a new PCR tube or a

96-well multiwell plate.

CRITICAL STEP: The volume of the master-mix may vary according to an activity of

restriction enzyme; units/µl). One unit of restriction enzyme will completely digest 1 µg

of substrate DNA in a 50 µl reaction in 60 minutes by definition. Enzyme activity is

usually dictated in the statement of commercially available restriction enzymes.

iii) Add 1 µl of restriction enzyme (5-10 units/µl) to the master-mix and mix the

reaction solution gently.

CRITICAL STEP: An important factor to achieve complete digestion with a restriction

enzyme is gentle mixing (pipetting the reaction mixture up and down or tapping the

reaction tube gently).

CRITICAL STEP: Enzymes should be stored at -20°C. While in use, enzymes should be

carefully maintained on ice.

8. Incubate reaction mixture (restriction enzyme and PCR product) on a PCR Thermal

Cycler or a Dry block heater at the optimal temperature (depending on restriction

enzyme) for appropriate times (2 hrs to overnight).

CRITICAL STEP: Generally 1 U of restriction enzyme digests 1 µg of DNA in 1 hour.

If an excess of enzyme is added to the reaction, the length of incubation time may be

decreased to save time. Conversely, a longer incubation time might be expected to

conserve on the amount of an expensive enzyme by using fewer units for the reaction.

Analysis of PCR-RFLP products by Gel Electrophoresis• TIMING 1 - 1.5 h

9. Load 6 µl (5 µl of each sample plus 1µl of gel-loading dye) of the amplified DNAs

digested by restriction enzymes (from step 8) onto either a 6%-12% polyacrylamide gel

or a 4% agarose (NuSieve 3:1) gel. Also load an optimal DNA size marker (e.g. 100 bp

ladder, pBR322-HaeIII, φx174-Hae III, or φx174-Hinf I) in a separate lane and in

parallel with samples on the gel.

CRITICAL STEP: Gel choice depends on user’s choice. Polyacrylamide gel is less

expensive than agarose gel, however requires more time and is more complicated to

make up.

10. Run the gel for appropriate times at constant voltage.

Running conditions depend on the respective gel. When electrophoresis is performed in

horizontal minigel electrophoresis apparatus (e.g. Mupid, ADVANCE Co. Ltd., Tokyo),

experimental conditions are 1 hr at 100 V for 12% polyacrylamide gel and 45 minutes at

100 V for 4% agarose gel.

11. Stain the gel with ethidium bromide at 0.5 µg/ml for 10 minutes.

12. Visualize products using a UV-transilluminator and a record the results by

photography. Check the cleaved fragments or undigested PCR product on the gel by

comparing with an optimal DNA size marker.

TIMING

Preparation DNA samples: 1- 24 h (depend on DNA extraction methods and samples)

PCR amplification of DNA samples (steps1-2): 1.5-2.5 h

Analysis of PCR products by gel electrophoresis (steps 3-6): 0.5-1 h

Restriction enzyme digestion of PCR products (steps7-8): 2 - 3 h

Analysis of PCR-RFLP products by gel electrophoresis (steps 9-12): 1-1.5 h

?Troubleshooting

If RFLP fragments are not observed as expected, then the unexpected outcome might be

due to many different reasons, such as e.g. (1) the digestion of PCR products is

inefficient, (2) the efficiency of the restriction enzyme reaction is poor or inactive, (3)

some restriction enzymes are inhibited by methylation35, 42 of nucleotides within their

recognition sequences, (4) star activity43 of some restriction enzymes, which cleave

similar but not identical sequences at other sites besides those containing the defined

recognition site. This altered or relaxed specificity is termed “star” activity. The star

activity is induced under certain extreme conditions (including high endonuclease

concentrations, high glycerol concentrations, low ionic strength, and high pH, and so

on). The following restriction endonucleases are reported to exhibit star activity, for

example, BamHI44, EcoRI45, MboII46, and SalI47.

When PCR products are incompletely digested or not digested at all, purification of the

PCR products with a commercial DNA extraction kit is sometimes useful for improving

or recovering the efficiency of the restriction enzyme activity. Increasing the number

of enzyme units added to the reaction mixture may also overcome the decreased

reaction efficiency associated with impure DNA preparation.

When a reaction of restriction enzyme appears to be incomplete, confirm the

inefficiency of the reaction by adding an internal control DNA which has cleavage site

with same restriction enzyme into the same tube.

Troubleshooting advice can be found in Table 1.

Table 1. Troubleshooting information.

Problems Possible Causes Solutions
Repeat amplification and ensure the cycling program.
Step 6 Change annealing temperature.
Incorrect thermal cycling program was used.
Presence of extra Change extension temperature.
amplification products Change cycling numbers.
Ensure that primer concentration is optimized.
Amplification mixture was not optimized. Decrease volume of the template DNA.
Change Mg2+ concentration.
Template DNA or Work area was contaminated. Eliminate all sources of contamination.
Taq polymerase was not added to Repeat amplification paying attention to the addition
Step 6 the amplification mix. and mixing of Taq with the amplification mix.
Absence of Taq polymerase was inactivated. Use new Taq or another Taq from different company.
PCR products Check whether all reagents were optimal, especially
Amplification mixture was not optimized.
template DNA concentration was optimal.
Check the cycler run history and ensure workings
Thermal cycler failure
of the thermal cycler.
Use new enzyme or
another enzyme from different company.
Step 12 Increase volume of enzyme.
Unexpected fragments Digestion was incomplete Dilute PCR product.
Perform digestion with longer incubation time.
Check reaction temperature.
remove PCR additives such as DMSO or glycerol.
PCR products were contaminated Purify PCR products with the DNA extraction kit.

ANTICIPATED RESULTS
PCR-RFLP is a relatively simple and inexpensive method of genotyping mainly single

nucleotide polymorphisms (SNPs). This method enables not only the discrimination of

homozygous and heterozygous samples at the target point mutation, but also the

genotyping of multiple mutations such as Human Leukocyte Antigen (HLA)

alleles22,32-33 (Figure 1). This method is especially convenient to confirm nucleotide

sequences at particular sites of interest without complex typing methods such as

hybridization, nucleotide sequencing DNA chips etc.

COMPETING INTERESTS STATEMENT

The authors declare that they have no competing financial interests.

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Figure 1

PCR-RFLP typing of HLA-DRB1 (see Ref. 32)

A. HLA alleles are determined with the combined patterns of restriction enzymes,

which have a single recognition site in some alleles but none in the amplified

regions of other alleles. Assignment of alleles is based on the combination of

band patterns produced from multiple restriction enzymes32,33. The gel picture

shows the typical restriction pattern obtained in the case of the

HLA-DRB1*0803 allele. The enzymes used in each lane, the sizes of the

products obtained and whether the enzyme cuts the product or not are detailed in

the box on the right.

B. The nucleotide sequence of the PCR-amplified exon 2 region of the DRB1*0803

allele is shown, with the cleavage sites for the restriction endonucleases detailed.

Primer sequences are highlighted in red.

Figure 2

ABO genotyping by PCR-RFLP (see ref. 34).

A. RFLP patterns of 5 DNA samples after digestion of the PCR-amplified products by

Kpn I and Alu I enzymes. Lanes 1-5 are samples with genotype OO, BO, AO, AO,

and BO, respectively. Their types are determined according to the restriction

patterns observed, as explained in Fig. 2b.

B. ABO genotype is assigned by intersection of possible genotypes from patterns of

 DNA fragments by Kpn I and Alu I digestion (upper table). ABO genotypes can

be determined for the 5 DNA samples shown in Fig. 2a (lower table).

C. ABO alleles can be determined by amplifying the ABO glycosyl transferase locus.

Exons 6 and 7 are amplified by PCR with 2 pairs of primers highlighted on the

figure by labeled coloured lines, primer1/primer 2 and primer 3/primer4,

respectively and digesting with KpnI an AluI enzymes; Nucleotide 258 bp (G) is

deleted in O allele (represented by a green triangle on the right hand diagram) which

creates the Kpn I site. Allele B is discriminated by the Alu I enzyme which

recognizes the G to A transition at 700 bp. The partial nucleotide sequence of the ‘A’

allele of the ABO gycosyl transferase locus is shown , highlighting the bases at 258

and 700 bp (in brackets) which allow discrimination of the ‘O’ and ‘B’ alleles,

respectively. The cleavage sites for the restriction endonucleases are also detailed

on the right.

Figure 3

The mismatch PCR-RFLP method for the detection of Y393N in the exon 9 of the

BCKDHA gene (see ref. 38).

A. A single nucleotide change (a T to A transversion at position 1324 in BCKDHA

gene, shown in pink in a yellow box in the sequence) causes a tyrosine to

asparagine substitution (Y393N). This substitution is discriminated using a

mismatch PCR-RFLP method; a PCR primer (5’-primer) was designed to

incorporate one mismatch with the original template sequence (shown in red) to

identify the Y393N substitution by creating a Sca I site (5’AGTACT3’) in the

absence of the substitution. As an internal control, a Sca I restriction site was

 incorporated in the 3’ primer. Therefore, all PCR products yield 170bp fragment

after digestion with Sca I.

B. Schematic electrophoretic pattern of RFLP after digestion with Sca I enzyme.

The Y393N BCKDHA allele is identified by RFLP pattern.

Figure 1PCR-RFLP typing of HLA-DRB1

1-A
1 2 3 4 5 6 7 8 9 10 1: marker,pBR322/HaeIII digestion
2: AvaII, uncut, 265 bp
3: Fok I, cut, 170 bp, 95 bp
267 bp 4: Kpn I, uncut, 265 bp
174 bp
124 bp 5: Hae II, cut, 159 bp, 106 bp
6: Cfr13I, cut, 201 bp, 63 bp, 1 bp
7: SfaNI, uncut, 265 bp
8: SacII, uncut, 265 bp
HLA-DRB1*0803
9: BsaJI, cut, 204 bp, 61 bp
10: ApaI, cut, 205 bp, 60 bp
1-B
Forward Primer 10 20 30 40

ACGTTTCTTGGAGTACTCTACGGGTGAGTGTTATTTCTTC
50 60 70 80
AATGGGACGGAGCGGGTGCGGTTCCTGGACAGATACTTCT
90 100 110 120
ATAACCAAGAGGAGTACGTGCGCTTCGACAGCGACGTGGG
130 140 150 HaeII 160
GGAGTACCGGGCGGTGACGGAGCTGGGGCGGCCTAGCGCC
170 180
FokI 190 200
GAGTACTGGAACAGCCAGAAGGACATCCTGGAAGACAGGC
Cfr13I BsaJI
210 220 230 240
GGGCCCTGGTGGACACCTACTGCAGACACAACTACGGGGT
ApaI 250 260
TGGTGAGAGCTTCACAGTGCAGCGG
Reverse Primer

Cleavage sites of restriction enzymes

HaeII : 5’···R GCGCY···3’ Cfr13I : 5’···G GNCC···3’ FokI : 3’···CCTAC(N)13 ···5’
BsaJI : 5’···C CNNGG···3’ ApaI : 5’···GGGCC C···3’
 Figure 2 ABO genotyping by PCR-RFLP
2-A 2-B
1 2 3 4 5 1 2 3 4 5

200
171
128

88

KpnI AluI
2-C
Primer 1 (258 bp)

KpnI
A,B:GGTGACC
exon intron Primer 2 O :GGT ACC
Primer 3 (700 bp)
(661)

AluI
(789) A,O : GGCT
Primer 4 B : AGCT
Figure 3 The mismatch PCR-RFLP for the detection of Y393N in the BCKDHA gene

3-A 23 bp Y393N (GenBank; Z14093 S49270)

Y438N (Swiss-Prot; 004973 in P12694)
5’-Primer AGTACT ScaI site
TAC Y(tyrosine)
CCACCTGCAGACCTACGGGGAGTAC
ccacctgcagacctacggggagcactac AAC N(Asparagine)
ccactggatcacttcgataagtgagacctgctca
gcccacccccacccatcctcagctaccccgagag
gtagccccactctaaggggagcagggggacctga
cagcacaccactgtcttccccagtcagctc
cctctaaaatactcagcggccagggc
GGAGATTTCATGAGTCGCCGGTCCCG
3’-Primer Mismatch position
ScaI site TCATGA
16 bp 5’ primer c T
3’ primer a C
3-B (1) (2) (3) (4) (5)

250 bp (1) Marker: 50 base-pair ladder

200 bp 186 bp
(2) PCR product without digestion
150 bp 170 bp
147 bp (3) N393N homozygosity
100 bp
(4) Y393N heterozygosity
50 bp (5) Y393Y homozygosity