Cat. No. DY62 / DY625

Instructor’s Laboratory ProtocolEducational Kit

Storage conditions pg. 5

EasyGenotypingITS-PCR Genotyping of bacterial strains by ribotyping

www.dnagdansk.com

Instructor’s Protocol

3

The aim of this exercise is to use ribotyping method (ITS-PCR) for interspecific and intraspecific differentiation of the supplied

bacterial strains (DNA of 6 different species, 3 isolates each).

EasyGenotyping ITS PCR

www.dnagdansk.com 4

1. Kitcontents EasyGenotypingITSPCR1exercise(DY62)

EasyGenotypingITSPCR5exercises(DY625)

10xreactionbuffer 6 tubes (10 reactions per tube) 6 tubes (50 reactions per tube)

50mMMgCl2 6 tubes (10 reactions per tube) 6 tubes (50 reactions per tube)

dNTPsmixture 6 tubes (10 reactions per tube) 6 tubes (50 reactions per tube)

L1primer 6 tubes (10 reactions per tube) 6 tubes (50 reactions per tube)

G1primer 6 tubes (10 reactions per tube) 6 tubes (50 reactions per tube)

PCR-gradewater 6 tubes (10 reactions per tube) 6 tubes (50 reactions per tube)

TaqNova DNApolymerase 6 tubes (10 reactions per tube) 6 tubes (50 reactions per tube)

BacterialDNADNA of 6 different species, 3 isolates each (3 reactions per tube)

DNA of 6 different species, 3 isolates each (15 reactions per tube)

Agarose 5 g 25 g

50xTAE buffer for agarose electrophoresis 50 ml 100 ml

Ready-to-use M100-1000DNALadder

1 tube (10 electrophoretic lanes)

1 tube(50 electrophoretic lanes)

6xGREEN DNA Gel Loading Buffer 0.5 ml 1 ml

Student’slaboratoryprotocols 6 pcs 6 pcs

Instructor’slaboratoryprotocol 1 pcs 1 pcs

I. Kitcontentsandstorage

5

2. Storageconditions

p Frequent freezing and thawing of all reagents should be avoided. p The PCR reagents should be stored at -20°C and thawed only before use. p The TaqNovaDNApolymerase should be stored at -20°C. p TemplateDNA should be stored at -20°C for long-term. After thawing,

samples can be kept at +4°C. p The MolecularWeightDNALadder may be stored at room temperature

or +4°C. If a longer storage period is required, a temperature of -20°C is recommended.

p The DNA Gel Loading Buffer (6xGREEN) may be stored at either room temperature or +4°C for up to 12 months.

3. Shippingconditions

p Reagents for PCR set up are shipped on dry or blue ice. p DNA templates are shipped on dry or blue ice. p DNA Ladder is shipped on dry or blue ice. p Other reagents for electrophoresis are shipped at room temperature.

www.dnagdansk.com 6

I. NOTESFORTHEINSTRUCTOR

1. Additionalequipmentandmaterialsrequired(notincludedinthekit)

p Agarose electrophoresis equipment (apparatus, power supply)

p PCR tubes (0.2 ml) p 1.5 ml Eppendorf tubes p Microcentrifuge p UV transilluminator or lamp p Thermal cycler

p Automatic pipettes with appropriate tips

p Microwave oven or laboratory burner

p Ethidium bromide solution or other stain for DNA visualization

p Nitrile gloves

2. Additionalnotesfortheinstructor

p We suggest conducting the laboratory exercise with twelve students working in pairs.

p One laboratory protocol is provided per pair. p The reagents are divided into 6 sets containing the reagents necessary to

carry out PCR reactions.

II. Notesfortheinstructor

7

p The aim is to perform a genetic typing of 18 supplied samples containing DNA of 6 different species of bacterial strains, using the ITS-PCR method. Each species is represented by 3 isolates – the first two numbered 1-2 are very closely related and belong to the same genotype; isolate 3 represents a distinct genotype (which was demonstrated with other methods of genetic typing). The first aim is to check whether a differentiation of analyzed strains at the level of species (interspecific differentiation) is possible with the ITS-PCR method or for different species various electrophoretic profiles will be obtained. Another aim of the experiment is to determine whether the discriminatory power of the method is sufficient to differentiate the analyzed isolates within a species (intraspecific differentiation) and to classify them into different genotype group (1, 2 – genotype A, 3 – genotype B). A table which allows arranging the analysis and grouping the isolates can be found on page 18. Each group of students should prepare reaction mixtures for 3 isolates belonging to one species.

p Each pair of students should prepare 4 reaction mixtures:• for the DNA of 3 isolates belonging to one species • Negative control

The reactions can be prepared either separately or by preparing a “Master Mix”. Table 1 in the laboratory protocols provides the fields for the relevant calculations.

www.dnagdansk.com 8

Instructor’s Protocol

3. Theoreticalintroduction

The rrn ribosomal operon is widely used in evolutionary studies of both prokaryotic and eukaryotic organisms. It is associated with a high degree of conservation of the sequence within this region. The use of sequence analysis of the subunit 16S rRNA (or 18S rRNA) has been used in the taxonomical studies of bacteria and eukaryotes. Within the RNA encoding sequences of a small and large subunit of a ribosomal operon, apart from conserved regions used to study a relationship between distant taxonomic groups, there are polymorphic regions which enable a differentiation within a family and species.

In prokaryotes the ribosomal operon contains sequences encoding the 16S, 23S and 5S rRNA subunits. These genes are separated with polymorphic regions of different sequence and size (“spacer” regions). Within a single genome there are usually several rrn operons, which demonstrate differences within their “spacer” regions. These regions are a perfect molecular target used in phylogenetic studies. In Escherichia coli there are 7 copies of the rrn operon on the chromosome, enterococci have 5-6 copies and a much larger number of operons can be found in Bacillus subtilis. Bacteria such as Mycoplasma, Mycobacterium and Halobacterium have one copy of the rrn operon in their genomes.

Figure 1. The diagram of the rrn ribosomal operon in prokaryotes (the buckles designated as A, B, C, D represent the regions analyzed in phylogenetic studies – the description in Figure 2).

rrs rrl rrf’

A

D

B C

’

S rRNA tRNA S rRNA S rRNA tRNA

Gene expression products

TerminatorsPromoters

9

In methods based on analysis of the sequences of the ribosomal genome the techniques such as hybridization, PCR technique, as well as sequencing and restriction analysis are used. A classification of these methods is shown in the below scheme.

RIBOTYPING

BasedonPCRtechniquesBasedon

hybridizationtechniques

1.Amplificationofparticularregionofrnnoperon:

A – variabl e region in 16S rDNA gene

B – variable region between 16S rDNA and 23S rDNA genes

C – region enclosing tRNA genes

D – region enclosing 16S rDNA and 23S rDNA and polymorphic fragment

2.PCRproductsrestrictionanalysis

3.Electrophoresis

4.VisualizationandUVdetection

1.Amplificationofvariableregionbetween16SrDNAand23SrDNAgenes

2.Electrophoresis

4.VisualizationandUVdetection

1.Amplificationof16SrDNAgene

2.SequencingofPCRproduct

1.Amplificationofvariableregionbetween16SrDNAand23SrDNAgenes

2.Electrophoresisandsouthernblotting

3.HybridizationwithrDNAprobe

ITS-PCR RFLPHYBRIDIZATION

PCR-RFLP Figure 2. Ribotyping – the techniques used for analysis.

Notes for the instructor

www.dnagdansk.com 10

Instructor’s Protocol

RFLP (Restriction Fragment Length Polymorphism) is also applied in ribotyping methods based on hybridization techniques. The restriction fragments of DNA produced in the digestion of the genomic DNA hybridize with the probes complementary to the unique sites within the rrn operon, usually it is a probe for the evolutionary conserved 16S rDNA region.

The PCR technique is used in ribotyping to amplify different regions within the rrn operon. The obtained PCR products are then subjected to electrophoretic separation, sequencing or RFLP restriction analysis. ITS-PCR is one of the most commonly used methods of ribotyping. In this method, a variable region between 16S rDNA and 23S rDNA is subjected to analysis. Amplification is carried out using universal primers complementary to highly conserved sequences which surround the variable region. They can be applied for different bacterial species. The differences in size and sequence of the variable region enable an intraspecific and/or interspecific differentiation of bacterial strains. As a result of the amplification, DNA fragments of about 200-1000 bp are obtained.

Sequencing of the 16S rDNA (or 18S rDNA) encoding fragment is most commonly used to identify both bacterial strains and eukaryotic organisms. Some databases are available, which contain deposited DNA sequences enabling a comparison of the sequencing results with the sequences comprised in the database (e.g. www.ncbi.nih.gov). On this basis it is possible to assign the analyzed strain to a particular family and species or determine its phylogenetic relationships.

11

Literature:1. M.A. Jensen, J.A. Webster, N. Straus: Rapid identification of bacteria on the basis of

polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms; Appl Environ

Microbiol., 1993 April, 59(4): 945–952.

2. B. Krawczyk, J. Kur: Diagnostyka Molekularna w mikrobiologii; Publishing of Gdańsk

University of Technology, 2008.

Literature:1. M.A. Jensen, J.A. Webster, N. Straus: Rapid identification of bacteria on the basis of

polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms; Appl Environ

Microbiol., 1993 April, 59(4): 945–952.

2. B. Krawczyk, J. Kur: Diagnostyka Molekularna w mikrobiologii; Publishing of Gdańsk

University of Technology, 2008.

Notes for the instructor

www.dnagdansk.com 12

II. LABORATORYEXCERCISES

IMPORTANT!!!

c Remember to centrifuge the samples with the reagents after thawing so that the entire mixture is located at the bottom of the tube!!!

c Before beginning to prepare the PCR, the hands should be washed and the workstation equipped with tips, tubes and gloves. 3% hydrogen peroxide solution should be used to disinfect the work surface and small items of laboratory equipment such as pipettes and so forth.

c The system’s extreme sensitivity means that it is highly susceptible to contamination. Disposable gloves should therefore be worn at all times.

c It is recommended that each of two stages of the determination, namely, the preparation of the PCR and the electrophoresis of the PCR products is carried out in a separate room. Special attention should be paid to PCR products from previous reactions since they represent the greatest danger of contamination.

c The use of autoclavable pipettes is the best solution for preparing the PCR. The pipettes should be autoclaved from time to time.

c Using separate pipettes is recommended for each stage of the determination, in other words, for preparing and portioning the master mix, adding the template and loading the PCR products onto the gel.

c The rule that only one tube may be open at any particular moment should be observed as this reduces the risk of contamination.

c Touching the edges of the tubes should be avoided.

III. Laboratoryexercises

13

1. ITS-PCRreaction

A. Preparationofthereactionmixtureforasinglesample.

The mixture components should be added to a 0.2 ml PCR tube in the order presented in the table below. Each pair should analyze 3 isolates of one species (Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Staphylococcus aureus, Enterococcus faecium).

Reagent Volume[µl]

MMixfor…tests

Sum[µl]

Tobeportioned

intoamountsof[µl]:

PCR–gradewater 29

10xreactionbuffer 5

MgCl2[50mM] 2

dNTPs[8mM] 5

L1primer[10µM] 3

G1primer[10µM] 3

TaqNovaDNApolymerase[2U/µl]

1

DNAtemplate–ANALYZEDSAMPLE

2

Finalvolume 50

Table 1. Composition of ITS-PCR reaction mixture for the analyzed sample.

www.dnagdansk.com 14

Instructor’s Protocol

B. PreparationofthereactionmixturefortheNegativecontrol.

For each series of analyses, a Negativecontrol should be performed in order to verify the correctness of work and the purity of the reagents for the purpose of excluding possible false positive results. The composition of the mixture is presented in Table 2.

Reagent Volume[µl]

PCR–gradewater 29

10xreactionbuffer 5

MgCl2[50mM] 2

dNTPs[8mM] 5

FORWARDprimer[10µM] 3

REVERSEprimer[10µM] 3

TaqNovaDNApolymerase[2U/µl] 1

PCR–gradewater 2

Finalvolume 50

Table 2. Composition of ITS-PCR mixture for the Negative control.

The 4 reaction mixtures listed above (3 samples + negative control) may be prepared simultaneously. In such a case, the Master Mix (MMix) should be prepared; the quantities of reagents used for one sample should be multiplied by 5 (the number of the samples + 1). Table 1 provides the fields for the relevant calculations to be made. The value obtained for the total volume of MMix should be divided by the number of the samples for which the MMix is calculated; in other words, for 5. The value obtained is the volume of the reaction mixture, which should be portioned into the specific tubes. It should be consistent with the sum of components calculated for the reaction mixture for 1 sample. Beforeportioning,thepreparedMMixshouldbemixedbypipettingorvortexing.

15

The tubes containing the prepared reaction mixtures should be placed in the thermal cycler and the temperature and time profile presented in Table 3 should be set.

Stage Temperature[°C] Time[s]

initialdenaturation 94 300

denaturation 94 30

25 c

ycle

s

primerannealing 56 30

elongation 72 60

finalelongation 72 300

cooling 10 ∞

Table 3. PCR cycling conditions.

2. DetectionofthePCRproducts

A. Exemplaryagarosegelpreparation

p The electrophoretic separation should be conducted in 2% agarose gel. p Prepare 1x TAE buffer working solution. In order to obtain a working solution,

dilute one part of the 50x TAE buffer stock solution in 49 parts of distilled water; for example, add 980 ml of distilled water for every 20 ml of the stock solution.

p Dissolve 1 g of agarose in 50 ml of 1x TAE buffer by heating the suspension in a microwave. Use caution when handling, as the temperature of the solution can be extremely high. Cool the solution to approximately 60°C.

c CAUTION!Fromthispointon,nitrilegloves,whichprovideprotectionfromethidiumbromide,acarcinogenicagent,mustbewornatalltimesduringallsubsequentprocedures.

Laboratory exercises

www.dnagdansk.com 16

Instructor’s Protocol

p Add 5 μl of 1 mg/ml ethidium bromide solution or other nucleic acid stain . p Mix well and carefully pour into a gel casting tray equipped with the

appropriately placed comb(s). p Take care not to leave any bubbles in the gel. Bubbles already in the gel

prior to pouring may be indicative of the gel’s being too cool. If this is the case, re-heat the gel.

p After gel solidification, remove the combs carefully, ensuring that wells stay intact. Place the gel in an agarose gel electrophoresis apparatus and add 1x TAE buffer to a level above the surface of the gel.

B. Agarosegelelectrophoresis

p 10 μl of each DNA sample should be pre-mixed thoroughly with 2 μl of 6xGREEN DNA Loading Buffer before applying in a gel well.

p The PCR products obtained should be applied to the gel wells with pipettes, as follows:1. 10 μl of M100-1000 ready-to-use DNA Ladder2. 2 μl of 6xGREEN DNA Loading Buffer and 10 μl of the PCR mixture

of the analyzedsample3. 2 μl of 6xGREEN DNA Loading Buffer and 10 μl of the PCR mixture

of the Negativecontrol

c CAUTION!Specialcareshouldbetakenwhileapplyingthesamplestothegelinordertoavoidanoverflowofthesamplesfromwelltowell,whichcouldproducefalseresults.

p The separation should be conducted for 30 – 60 min, with a voltage of ca. 5 – 10 V/cm of gel length.

p Once the electrophoresis has been completed, the gel should be analyzed in the UV light of the transilluminator.

17

3. InterpretationofthePCRresults

Analysisofobtainedelectropherogram

p The agarose electrophoresis in the lane in which the separation of the DNA size marker (M100 – 1000) is performed should produce an electrophoretic profile consisting of 10 bands (100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 bp; Photo 1: Lane M).

p No signals should be obtained as the result of the electrophoresis in the lane in which the separation of the PCR reaction mixture of the Negativecontrolis performed. This is indicative of the correct preparation of the mixture and good reagent quality. Products below 100 bp, which may occur in the lane, are derived from primer interactions and are not taken under analysis. The presence of products above 100 bp indicates reagent contamination. The reaction should be set up again and analyzed as before (Photo 1: Lane K-). In the lane containing the analyzedsample, there should be a strain characteristic electrophoretic profile (Photo 1: Lanes 1 – 3), consists of bands of different size. These bands correspond to different variants of ITS region in rrn operon in particular species.

p The analysis of affinities of the test strains should be made by comparing the electrophoretic profiles obtained for the different samples (Photo 1: Lanes 1 – 3) and by completing Table 4 on the basis of the number and sizes of the PCR products. Strains should be assigned to one genotyping group if they have the same electrophoretic profile (in example A, Photo 1: Lanes 1 and 3). Strains should be assigned to separate genotyping groups if they have a different electrophoretic profiles (Photo 1. Lanes 1 and 3

– genotype A; lane 2 – genotype B). p In order to answer the question: “Is the ITS-PCR method suitable for

interspecific differentiation?”, it should be examined whether the isolates belonging to different species produce different (interspecific differentiation is possible) or the same (interspecific differentiation is not possible) electrophoretic profiles.

Photo 1. Example of an electropherogram with ITS PCR results.

Laboratory exercises

www.dnagdansk.com 18

Instructor’s Protocol

p In order to answer the question: “Is it possible to perform an intraspecific differentiation using the ITS-PCR method?” the profiles obtained for different isolates of the same species should be compared. Where different profiles are obtained, the isolates should be grouped and classified into different genotype groups. No differences in the electrophoretic profiles will testify about low discrimination potential of the ITS-PCR method and its inadequacy for intraspecific differentiation of given strains.

IsolateNo Genotype(e.g.A,B,C,...) IsolateNo Genotype(e.g.A,B,C,...)

Escherichia coli Serratia marcescens

E1 Sm1

E2 Sm2

E3 Sm3

Klebsiella pneumoniae Staphylococcus aureus

Kp1 Sa1

Kp2 Sa2

Kp3 Sa3

Klebsiella oxytoca Enterococcus faecium

Ko1 Ef1

Ko2 Ef2

Ko3 Ef3

Table 4. Results of ITS-PCR typing – classification to individual genotyping groups (genotypes).

EasyGenotyping ITS PCR

www.dnagdansk.com

BLI

RT

S.A

., ul

. Trz

y Li

py 3

/1.3

8, 8

0–1

72 G

dańs

k, w

ww

.dna

gdan

sk.c

omT:

+4

8 5

8 7

39 6

1 5

0 F

: +4

8 5

8 7

39 6

1 51

E:

info

@dn

agda

nsk.

com

WARRANTYThe kit components will remain stable for 12 months from the date of purchase providing it is stored properly. The electrophoresis buffers will remain stable for 6 months.