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The Vectorette System

"Gene Walking Made Easy"

INSTRUCTION MANUAL

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Technical support: 800 234 5362 [email protected] www.genosys.com

THE VECTORETTE MANUAL PAGE

1. THE VECTORETTE MANUAL 4

2. PURPOSE 4

2.1 WHAT IS VECTORETTE?2.2 PRINCIPLE OF ACTION2.3 FEATURES OF VECTORETTE2.4 APPLICATIONS FOR VECTORETTE2.5 EXAMPLES2.6 ADVANTAGES

3. PROTOCOLS 11

3.1 STEPS3.2 BEFORE YOU START

3.2.1 Restriction Enzyme Digestion3.2.2 Ligation3.2.3 Vectorette PCR3.2.4 Nested PCR3.2.5 YAC DNA3.2.6 YAC PCR

3.3 TROUBLESHOOTING

4. CLONING OF VECTORETTE PCR PRODUCTS 19

5. SEQUENCING OF VECTORETTE PCR PRODUCTS 20

5.1 LAMBDA EXONUCLEASE METHOD5.2 DNA SEQUENCING

6. VECTORETTE PRODUCTS AVAILABLE FROM 22

SIGMA-GENOSYS

7. REFERENCES 24

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8. APPENDICES 25

8.1 GLOSSARY OF TERMS8.2 BUFFERS

FIGURES

1. Schematic of Vectorette design 5

2-5. Examples of Vectorette applications 10

6. Restriction enzyme sites in Vectorette II 20

Vectorette is a trademark of Sigma-Genosys

This product is designed and sold for use in the Polymerase Chain Reaction (PCR) process covered by

patents owned by Hoffman-La Roche. Use of the PCR process requires a license. A license for research may

be obtained by purchase and use of both authorised reagents and DNA thermal cyclers from the Perkin-

Elmer Corporation or by otherwise negotiating a license with Perkin-Elmer.

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1. THE VECTORETTE MANUAL

This manual contains all the information you should need to be able to use the Vectorette

system. We recommend that before starting your experiments, that you read the

relevant sections of the manual. All references to Vectorette in this manual are for

Vectorette II, which was designed to function similarly to the original Vectorette I, but

contains common restriction enzyme sites in the mismatched region. The restrictions

sites were included to facilitate cloning of the final PCR product, if desired. Sigma-

Genosys currently sells and stocks Vectorette II only.

2. PURPOSE

2.1 WHAT IS VECTORETTE?

The polymerase chain reaction (PCR) has revolutionised molecular biology since its

original description in 1985 (Saiki et. al. 1985). The ability to amplify specific fragments

of DNA has found a wide range of applications in all aspects of life science research.

Typically, PCR requires the prior knowledge of the DNA sequence of two different primers

at either end of the fragment to be amplified. For a large number of applications it would

be useful to be able to amplify DNA fragments where the sequence of only one end is

known. Vectorette PCR is a method for performing this feat, allowing amplification of

any uncharacterized sequence adjacent to a known region (Lilleberg et. al. 1998).

Vectorette PCR was invented and patented in 1988 and has since been used as a tool for

intense research and development. The technique has been optimized for a number of

different applications and the DNA sequences used in all the Vectorette products have

been checked against Genbank to ensure minimum cross-homology problems for any

target DNA sequence to be amplified.

2.2 PRINCIPLE OF VECTORETTE SYSTEM

Vectorette units are specially designed double stranded DNA fragments that have a

stretch of mismatched nucleotides in the middle (Figure 1, below). A range of Vectorette

units are available that are designed with appropriate overhang at one end to ligate to

DNA fragments generated by a range of different restriction enzymes, in addition to blunt

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ligations (see sections 2.6 and 6). This allows a wide flexibility in the choice of PCR

products generated from a particular locus.

Figure 1. Diagram to show the Vectorette system process.

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The Vectorette system consists of three key steps as seen in Figure 1:

(i) Digestion of sample DNA with a restriction enzyme

(ii) Ligation of appropriate Vectorette units to the restriction digested DNA

fragments, generating a Vectorette library.

(iii) PCR using one primer directed at the Vectorette unit and a custom primer

targeting the known DNA sequence (the initiating primer). This allows PCR of a

fragment of DNA between the known sequence and the restriction site used to

cut the target DNA.

A custom primer initiated from known genomic sequences adjacent to an unknown

region is directed towards the ligated Vectorette end. The Vectorette primer is identical

to the bottom strand of Vectorette in the mismatched portion (Figure 1, inset).

Therefore, the Vectorette PCR primer has no complementary strand to anneal to in the

first cycle of PCR. The initiating primer from the known sequence (directed towards the

sequence of interest) will produce a complementary strand to the bottom strand of the

Vectorette in the first cycle of PCR. In the second cycle of PCR there is now a template

for the Vectorette PCR primer. This template contains the initiating primer from the

known sequence at the other end to the Vectorette sequence. After the second cycle,

PCR continues normally to amplify the targeted sequence (Figure 1).

In some gene walking projects, the distance between the known sequence and the

restriction site is not known. Hence it is advisable to make several libraries with different

restriction enzymes to ensure amplification of an optimum sized PCR fragment as well as

differing sizes of PCR products containing the region of interest. The longer the PCR

product, the farther away the custom primer sequences is from the restriction site.

Sequences from the restriction site end of the longest PCR fragment can be used to

design another primer for the next step.

2.3 FEATURES OF VECTORETTE

The basic design of the Vectorette unit and the location of the PCR and sequencing

primers are shown in Figure 1. The essential features of the design are:

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1) Restriction fragment compatible end.

There are five different Vectorette ends available:

a) Bam HI (also compatible with Bgl II, Bcl I and Sau 3A)

b) Cla I (also compatible with Mae II, Hpa II and Taq I)

c) Eco RI

d) Hind III

e) Blunt (e.g. Alu I, Eco RV, Hae III, Pvu II, Rsa I, Sma I).

One feature of the sequence at the end of the Vectorette is that although it is

ligatable to a particular sticky/blunt end, the original restriction site is not

reformed in the target-Vectorette construct (N.B. This is not the case for two

particular four-base cutters; Sau 3a/Mbo I and Hpa II/Msp I).

e.g. Vectorette Eco RI end = 5' -- AATT'G --- 3' 3’ C---5’

Therefore in ligated construct = 5’— GAATTG--3’ 3’- CTTAAC—5’

This is not cut by Eco RI.

This means that during Vectorette library construction, there is no need to heat

inactivate the restriction enzyme before the ligation step (except for the enzymes

mentioned above). A consequence of this is that ligated “target-target” molecules

will be cut by the restriction enzyme, but the “target-Vectorette” ligations will not

be cut. This helps to increase the overall yield of target molecules, which become

ligated to Vectorette ends.

2) The mismatched region

The unpaired region of the Vectorette unit is crucial to its function. The Vectorette

PCR primer has the same sequence as part of the bottom strand of the

mismatched region. The PCR primer has no complementary sequence to anneal to

and therefore, cannot prime DNA synthesis during the first cycle of PCR. The

complement can only be generated by extension from an initiating primer (a

custom primer directed at the “known” sequence), transcribing through to the

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Vectorette end. Therefore, the only PCR products formed are those which contain

the initiating primer. The bottom strand also contains restrictions sites for Bam

H1, Eco R1 and Hind III that could facilitate cloning of the final PCR product if

necessary.

3) The completely paired regions

These regions are exactly complementary to each other and provide the double-stranded

region of the Vectorette unit. The double-stranded regions the Vectorette stabilize the

unit, forming a partially double-stranded molecule in solution.

2.4 APPLICATIONS

The use of Vectorette technology enabling isolation, amplification and analysis of novel

DNA sequence adjacent to a known sequence makes it suitable for a wide range of

potential applications. These include:

1) Genome walking.

2) DNA Sequencing of Yeast Artificial Chromosomes (YAC) insert

termini.

3) DNA sequencing of the termini cosmid inserts.

4) Mapping of promoters and/or introns in genomic DNA using cDNA

sequences.

5) Sequencing of large clones without sub-cloning.

6) Mapping of regions containing deletions, insertions, translocations

etc.

7) Gap-filling in genome mapping projects.

2.5 EXAMPLES

This section describes some specific examples of the different applications of the

Vectorette system.

• Genomic walking in bacterial genomes

Several genomic walks were initiated in the human pathogenic bacteria Chlamydia

trachomatis from existing characterized sequence, in order to determine new regions

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with potential applications as diagnostic probes. New primers were designed by

sequencing the termini of the Vectorette PCR product to enable further steps to be

taken. Figure 2 shows a PCR experiment with Vectorette PCR from a known primer at the

5' end of the Chlamydia trachomatis 16S rRNA gene. Note that no sequence information

downstream of the known primer is needed in order to amplify these segments of DNA.

The distance between the known primer and the next restriction site for a particular

enzyme determines the length of the amplicons.

• Genomic walking in human genomic DNA.

Figure 3 shows amplification of human genomic DNA from the KM19 locus. DNA digested

with Eco R1 and ligated to Vectorette ends. Vectorette PCR has been used to walk

human genomic DNA from known start points.

• Sequencing of YAC insert termini.

Yeast artificial chromosomes (YACs) allow the cloning of large fragments of DNA (100-

500 Kb). However, large fragments of DNA are difficult to characterize. The Vectorette

system has been used to walk from the YAC arms into the unknown cloned region,

allowing the sequence at either end of the insert to be determined. This is useful in

identifying overlapping YAC clones, in mapping of the inserts and in ordering a contig of

YAC clones (see Figure 4).

• Amplification of Chlamydia

Vectorette DNA digested with Cla I enzyme from the known sequences of the momP

gene to the Vectorette ends (Figure 5).

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2.6 ADVANTAGES

Vectorette could be used for an entire sequencing project without the need for sub-

cloning. Some of the advantages of the Vectorette system over existing technologies

are:

i) Cell-free gene manipulation. Vectorette PCR can effectively replace cloning and

sub-cloning in many molecular genetics projects. This saves time, avoids safety

problems due to gene manipulation legal requirements, and also avoids problems

associated with unclonable sequences or ones that are highly unstable in particular

host/vector systems.

ii) Can be used with limiting amounts of starting material. Vectorette system is

based on PCR, only small quantities of DNA are needed e.g. 100 ng of bacterial

DNA or 1 µg of human genomic DNA.

iii) Do not need high purity DNA. If the target DNA can be digested by restriction

enzymes, then it will be pure enough for Vectorette. As Vectorette uses the

specificity of PCR, contamination of target DNA with other DNA sources is usually

not a problem.

3. PROTOCOLS

3.1 BEFORE YOU START

Vectorette can be used for a wide variety of applications. Each application has different

requirements, which should be considered carefully before starting your experiments. All

Vectorette units and primers are shipped lyophilized. Each Vectorette II unit tube

contains 15 pmol of lyophilised product to be resuspended in 25 µl of sterile distilled

water to achieve the recommended concentration of 0.6 pmol/µl. The amount of

lyophilised Vectorette II primer and nested primer in each tube is 10,000 pmol. This is to

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be resuspended in 100µl of sterile distilled water to obtain a recommended concentration

of 100 pmol/µl. Below are given some general guidelines.

Ø Choice of restriction enzyme

The available Vectorette units allow the user the choice of either a six-base cutter or a

four-base cutter. For applications where the maximum amount of uncharacterized

sequence information is desired e.g. genome walking, then a selection of six-cutters is

recommended. For other applications where the length of the PCR product obtained is

not critical e.g. characterizing the ends of YAC inserts, then one or more four-cutters will

be sufficient. Another consideration in the choice of restriction enzyme depends on the

source of target DNA. High G+C content genomes have a correspondingly higher rate of

methylation. Care must be taken to choose restriction enzymes that cut at unmethylated

sites.

Ø Amount of template

The amount of DNA template used to construct a Vectorette library will vary according to

the species and the genome size. Another consideration is the number of restriction

sites in the target DNA i.e., 4-base cutters cut more frequently than 6-base cutters and

therefore more ends are generated during digestion. As a rough guide, a 1.5 Molar

excess of Vectorette units to target DNA ends is the minimum requirement needed for

successful Vectorette library construction.

Ø Custom gene specific primer

The initiating primer complementary to the known DNA sequences provides specificity for

the Vectorette-PCR reaction and requires careful design consideration. Unlike

conventional PCR, the gene specific primer provides the only source of specificity to

ensure faithful amplification in Vectorette PCR. Therefore care must be taken to ensure

“uniqueness” of primer design and to avoid annealing to sequences bearing homology to

repeat sequences, e.g. CA repeats in higher organisms. It is recommended that initiating

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primers are designed with a Tm in the range of 65-75 0C. Avoid hairpin structures and

self-complementary 3’ ends.

3.2 STEPS

There are three basic steps in producing a Vectorette PCR product, all of which are

commonly used molecular biology techniques:

1. Restriction Enzyme Digestion,

2. Ligation of Vectorettes to digested target DNA

3. Vectorette PCR.

The first two steps (Restriction Enzyme Digestion and Ligation) are basic molecular

biology techniques and can be carried out very simply according to the generalized

protocol below. The wide variety of applications in which PCR is being used, make it

difficult to describe a single set of conditions that will guarantee success. A generalized

protocol for PCR is given below which has been shown to work well with Vectorette

libraries constructed as described below. Users may need to modify these protocols

depending on their application.

3.2.1 Restriction Enzyme Digestion

This is a generalized protocol. Please modify this protocol according to your experimental

conditions or recommendations form your restriction enzyme supplier..

i) Combine the following in a sterile microcentrifuge tube:

DNA in water or 1X TE buffer (As a rough reference: 100 ng bacterial DNA or

1-2 µg Human Genomic DNA)

5 µl 10 x Restriction Buffer

10-20 units Restriction Enzyme

Sterile Deionized Water to 50 µl

ii) Incubate the sample at 37°C for 1-2 hour.

ii) Monitor digestion of fragments by agarose gel electrophoresis

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3.2.2 Ligation

Ligate 5 µl of the corresponding Vectorette units to the digested end of the DNA sample

using 1 unit of T4 DNA ligase. ATP and DTT are added to a final concentration of

approximately 2mM. This reaction can be carried out in the same tube as the restriction

digest without modification. Most restriction buffers are compatible with that for T4 DNA

ligase.

i) To the microcentrifuge tube containing the restriction digest add:

5 µl Vectorette units (3 pmol)

1 µl 100 mM ATP

1 µl 100 mM DTT

1 unit T4 DNA ligase

ii) Incubate the microcentrifuge tube at 20°C for 60 minutes followed by 37°C for

30 minutes. Repeat this incubation procedure three times

Note: The reason for repeat temperature cycling is to re-digest any target DNA

fragments which have ligated to each other and not to Vectorette units. The

cycling therefore ensures optimum ligation of Vectorettes to target ends. If

you are using a 4-base cutting restriction enzyme whose site is reformed on

ligation, i.e. Sau 3A, Msp I, do not repeat the incubation procedure. Instead

leave at room temperature for 4 –5 hrs after the ligation incubation. When

using these 4-base the restriction digest must be heat denatured (65-70°C for

10-15 minutes) before ligation.

iii) After incubation add 200 µl of sterile water and store the Vectorette library

in small aliquots (approximately 20 µl) at -20°C.

3.2.3 Vectorette PCR

The wide variety of applications in which PCR is being used make it difficult to describe a

single set of conditions that will guarantee success. Detailed below is a basic PCR

protocol. However you may need to modify the conditions for optimum results.

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For a 100 µl PCR reaction:

3) Combine the following in a sterile microcentrifuge tube:

1 µl of Vectorette library

10 µl of 10x PCR buffer (optimize the Mg2+ conc. for the reaction)

50 µM of each dNTPs (final concentration)

100 pmol of Universal Vectorette Primer

100 pmol custom initiating primer

1µl of Taq DNA polymerase (5U/µl)

This PCR cycle requires the use of a hot start for high specificity. This could be achieved

by using the JumpstartTM Taq DNA polymerase (Sigma-Aldrich, St. Louis, MO).

94°C - 1 minute

55-65°C - 1 minute (Temp. depends on the annealing temperature of specific primer)

72°C - 1-3 minutes

----------------------------------

35-40 cycles

Appropriate controls for this experiment include a reaction with no template, one with

the Vectorette PCR primer only, another with gene specific custom primer only. For

obtaining longer PCR products (above 2 kb), we recommend using AccuTaqTM LA DNA

Polymerase in combination with TaqStart Antibody for hot start. The extension times at

72°C should be regulated according to the length of the amplicon. As a rough estimate:

1000 base pairs for each minute. In cases where adequate specificity or yield is not

obtained we recommend using a “touchdown” PCR thermal cycler profile:

STEP TEMP. TIME

1 96°C 1 min

2 94°C 40 sec

3 70°C 40 sec continued…

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reduce temp. of step 3 by 0.50C per cycle

4 GO TO STEP 2 19 TIMES

5 92°C 40 sec

6 60°C 40 sec

increase time of step 6 by 1 sec per cycle

7 GO TO STEP 5 19 TIMES

ii) Analyze PCR products on a 1.5% - 4% agarose gel, depending on the size of the

amplified fragment. With complex DNA targets, it is common that faint or no

bands may be seen after the initial PCR. However, discreet products may appear

following a subsequent nested PCR (see next section).

3.2.4 Nested PCR

For higher specificity it is recommended that a nested custom primer also be used. The

nested primer should be extended at the 3’end by 3-5 bases, relative to the custom

initial primer. The nested Vectorette PCR primer is included in the kit. It may be

necessary to optimize the Mg2+ conc. for the reaction.

Standard guidelines for the second round of PCR:

For a 100 µl reaction:

1 µl (of a 1:1 to 1:10,000 dilution) of the primary PCR product

l0 µl of 10x PCR buffer (with optimum Mg2+ conc.)

50 µM of each dNTPs (final concentration)

100 pmol of nested Vectorette Primer

100pmol of nested custom primer

Use similar PCR procedures and control experiments as described in the previous section

(3.2.3.i). Run the nested PCR product on a 1.5–4% agarose gel.

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3.2.5 Yeast Artificial Chromosome DNA ('YAC' DNA)

General protocols for the handling of YAC clones, preparation of agarose plugs can be

found in (Anand et. al. 1990). YAC DNA is stored in agarose plugs, therefore the

restriction and ligation steps involved in Vectorette library preparation can be carried out

without ethanol precipitation or column purification of the DNA.

i) Prior to digestion, equilibrate 1 µg of YAC DNA (1/3 of a plug) in 1 ml of cold

TE buffer overnight (TE = 10 mM Tris-Cl pH 7.6, 1 mM EDTA).

ii) Replace the TE buffer with 1 ml of the relevant 1 x Restriction Buffer for 1

hour on ice.

iii) Replace the buffer after 1 hour with 100µl of fresh 1 x Restriction buffer and

20 units of the chosen restriction enzyme. Incubate overnight at 37°C.

iv) Replace the restriction buffer/enzyme solution with 1 ml of 1 x ligase buffer

(50 mM Tris-Cl pH 7.6, 10 mM MgCl2 , 1 mM DTT and equilibrate on ice for

1 hour.

v) Replace the 1 x ligase buffer with 100 µl of fresh ligase buffer and add 5 µl

of the appropriate Vectorette units (3 pmol).

vi) Melt the plug at 65°C for 10 minutes, followed by cooling to 37°C.

vii) Add ATP to 1 mM and add 1-10 units of T4 DNA ligase and incubate at

37°C for 2 hours.

viii) Add 100µl of sterile water and store in aliquots at -20°C.

3.2.6 YAC Vectorette PCR

YAC Vectorette PCR is the same as normal Vectorette PCR, as described previously in

section (3.2.3.i).

3.3 TROUBLESHOOTING

There is only one step by which the success of a Vectorette library preparation can be

gauged. The indication of success is the presence of an ethidium-stained band of the

correct size, after running the PCR products on an agarose gel. The following list gives

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remedies to symptoms based around the presence/absence of a Vectorette PCR product

band.

SYMPTOM POSSIBLE CAUSE APPROPRIATE ACTION

No band Sample DNA impure or degraded Re-purify or replace DNA

Essential component missing Ensure correct addition of components

Loss of enzyme activity Replace enzyme

Lack of specificity (see below)

DNA fragment too large Modify PCR conditions (see section 3.3.3i)

or change restriction enzyme

Smear Lack of specificity (see below)

Multiple Lack of specificity (see below)

bands Partial digest Increase amount of functional enzyme and/or

Extend digestion time. Alternatively, add 10mM of

spermidine to reaction.

Below, we give some suggestions for optimizing Vectorette PCR reactions in order to

increase the specificity of the reaction.

1. Increase annealing temperature during PCR. Alternately, try the touchdown protocol

that has been included in this manual. Use “hot start” protocols.

2. Use thin wall tubes and minimize the incubation time during the annealing and

extension steps. This will limit the opportunities for mispriming and extension. It may

be necessary to reduce annealing times to 15-30 seconds.

3. Ensure the primers and enzyme concentrations are not too high, this will help reduce

mispriming.

4. Magnesium ion concentrations also play an important role in specificity. Changing

the magnesium ion levels can improve specificity (and perhaps yield) by increasing

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the stringency of the reaction or by direct effects on the polymerase itself. The

magnesium ion : dNTP ratio also plays a role in PCR specificity.

Important note:

For certain applications (particularly when using complex target DNA), nested PCR

is required for complete specificity.

4. CLONING OF VECTORETTE II PCR PRODUCTS

The current version of Vectorette (VectoretteTM II) was designed for researchers who

wish to clone their Vectorette-PCR products in a particular orientation. The Vectorette-

PCR products contain the recognition sites for three different restriction enzymes (Bam

HI, Eco RI, Hind III) (see Fig 6). The restriction enzyme recognition sequences are

single-stranded in the Vectorette unit, and therefore do not affect Vectorette library

construction. However the recognition sequences become double-stranded during

Vectorette PCR so that Vectorette - PCR products contain these restriction sites. The

rationale behind choosing these three particular enzymes was to reduce the possibility of

cutting elsewhere in a Vectorette II PCR product. If a Vectorette library is constructed

with one of these enzymes then the same enzyme will cut only in the Vectorette part of

the amplicon.

A phenol/chloroform extraction followed by ethanol precipitation of PCR products is

recommended prior to digestion. One potential concern is that two different fragments,

each with a sticky end and a blunt end will be produced: the main Vectorette PCR

product containing the sequence of interest and a short (15-30 bp) fragment derived

entirely from the Vectorette unit. To avoid cloning the smaller fragment, the larger

fragment can be purified from agarose after restriction digest and electrophoresis.

Another method is to create a sticky end at the gene specific end of the PCR product

(different from sticky end at the Vectorette end) by using a gene specific primer

containing an infrequent restriction site at the 5’ end. This will reduce the possibility of

cutting within the “unknown” amplified regions. Detailed cloning protocols can be found

in Sambrook et. al. (1989) and other molecular biology protocol books.

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5. SEQUENCING OF VECTORETTE PRODUCTS

Vectorette PCR products may be sequenced by two different methods:

1) Direct sequencing of PCR products by dideoxy methods.

2) Sub-cloning of PCR into an appropriate vector, followed by a suitable

sequencing method.

The PCR product can be purified by using spin columns such as GenElute kit (Cat.# GEN-

PCR, Sigma-Aldrich, St. Louis , MO) to remove excess primers, dNTPs and salts. Single

stranded DNA templates for sequencing can be made by lambda (λ) exonuclease

treatment, from amplicons made with one phosphorylated and one unphosphorylated

primer. λ-exonuclease has a 5’→3’ exonuclease activity and preferentially digests DNA

containing a 5’-phosphate group. A protocol for generating single stranded templates by

λ-exonuclease method is given below. Sigma-Genosys offers both phosphorylated and

unphosphorylated Vectorette primers.

BamH1

Eco R1

Hind III

Restriction Sites

VECTORETTE

Bottom

Figure 6

Top strand

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5.1 LAMBDA EXONUCLEASE METHOD

Purified PCR products are checked by agarose electrophoresis to ensure single band

amplification in each case.

1) Add an equal volume of TE buffer equilibrated phenol to PCR reactions and

vortex for 5 seconds.

2) Spin in a microcentrifuge for 5 minutes.

3) Transfer the upper aqueous layer to a 1.5 ml sterile microcentrifuge tube.

4) Add two volumes of ethanol, pre-cooled at -20°C. Mix thoroughly and chill

the sample on dry ice or at –200C for 15 minutes.

5) Spin the sample in a microcentrifuge for 10 minutes.

6) Remove the supernatant carefully, ensuring that the DNA pellet is

undisturbed.

7) Wash pellet with 70% ethanol.

8) Dry the pellet in a SpeedvacTM concentrator for 10 minutes.

8) Redissolve the dried pellet of DNA in 20 µl of sterile distilled water.

9) To the purified PCR product, add the following:

2.5 µl 10 x λ-exonuclease buffer (67 mM glycine-NaOH pH 9.4, 2.5 mM

MgCl2)

1.5 µl sterile water

1 µl λ-exonuclease (4 units/µl)

10) Mix gently by hand. Incubate at 37°C for 30 minutes.

11) Add 25 µl of sterile water to the tube and perform two phenol extractions as

described in steps 2-4.

12) To the extracted aqueous layer add 17 µl of 3M NaOAc and 170 µl of pre-

cooled ethanol. Mix thoroughly and chill for 15 min. at –20°C.

13) Spin at maximum speed in a microcentrifuge for 10 minutes.

14) Using a pipette, carefully remove the supernatant ensuring that the DNA

pellet is undisturbed.

15) Dry the pellet in a SpeedvacTM concentrator for 10 minutes.

16) Redissolve the dried DNA pellet in 20 µl of sterile water.

17) The template is now ready to be sequenced.

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5.2 DNA SEQUENCING

Protocols for manual or automated DNA sequencing can be obtained from several

companies including those from Amersham Pharmacia Biotech

(http://www.apbiotech.com/technical/technical_index.html), PE Biosystems (http://www2.perkin-elmer.com/ab/),

Licor, Inc. (http://www.licor.com). The amount of Vectorette sequencing primer (15mer

and 20mer) is 500 pmol of lyophilised product. Resuspend in 50-100µl of sterile distilled

water to obtain the appropriate concentration according to the sequencing protocol

followed.

6. VECTORETTE PRODUCTS AVAILABLE FROM SIGMA-GENOSYS

Sigma-Genosys provides a full range of Vectorette products, which allows the user

freedom and flexibility in the approach to their particular application.

There are two versions of the starter pack containing either the 15-mer or the 20-mer

sequencing primer for manual or automated sequencing methodologies respectively.

Vectorette II Starter Pack S contents: (Catalog#DN-16-010A)

1) Five different Vectorette ends (Bam HI, Cla I, Eco RI, Hind III and blunt). Each unit

contains 15 pmol of lyophilised product. The recommended concentration in sterile

water is 0.6 pmol/µl.

2) Vectorette primer and nested prime for PCR applications. Both primers are available

with or without a 5’-phophorylated end. Amount of each primer per tube is 10nmol.

The Tm of the unphosphorylated primer is 71.9°C. Tm of nested unphosphorylated

primer is 73.0°C.

3) 15-mer sequencing primers . Amount of primer per tube is 500 pmol. The Tm of the

15-mer is 55.1°C.

Vectorette II Starter Pack T (Cat# DN-16-020A) is same as Starter Pack S except that it

contains the longer 20-mer sequencing primer which is more suited for automated

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sequencing. Tm of the 20-mer is 65.4°C. PCR and sequencing primers for pYAC4

subcloning and sequencing OF YAC insert termini using Vectorette are also available.

The components of the Vectorette system are available as separate items for reordering

and have been listed below along with the catalog numbers. Each is Vectorette unit

contains sufficient material for 5 ligations, each PCR and sequencing primer contains

sufficient material for 100 reactions.

Product Catalog number

§ EcoR1 Vectorette II DN-16-210

§ HindIII Vectorette II DN-16-220

§ BamHI Vectorette II DN-16-230

§ ClaI Vectorette II DN-16-240

§ Blunt End Vectorette II- DN-16-250

§ Vectorette II Primer (phosphorylated) DN-16-320

§ Vectorette II Primer (unphosphorylated) DN-16-325

§ Vectorette II Nested Primer (Phos.) DN-16-360

§ Vectorette II Nested Primer (Unphos.) DN-16-365

§ pYAC4 Right Arm Primer (Phos.) DN-16-340

§ pYAC4 Right Arm Primer (Unphos.) DN-16-345

§ pYAC4 Left Arm Primer (Phos.) DN-16-350

§ pYAC4 Left Arm Primer (Unphos.) DN-16-355

§ Vectorette II Sequencing Primer (15mer) DN16-420

§ Vectorette II Sequencing Primer (20mer) DN-16-430

§ Vectorette II Sequencing Primer (20mer; Phos) DN-431

§ Pyac4 Right Arm Sequencing Primer DN-16-440

§ Pyac4Left Arm Sequencing Primer DN-16-450

Also available:

i) Vectorette II starter packs, containing Vectorette units for all five restriction ends,

as well as phoshorylated Vectorette PCR primer and sequencing primer.

ii) YAC PCR primers and sequencing primers to allow the sequencing of YAC insert

termini using Vectorettes.

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7. REFERENCES

Lilleberg, S. and S. Patel. Isolation of DNA flanking retroviral integration sites using

Vectorette II . Genosys Origins, Vol I, No. II. 1998.

Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B.

and H. A. Erlich. Primer-directed enzymatic amplification of DNA with a

thermostable DNA polymerase. Science 239 (4839): 487-491. 1988.

Anand, R. and J. Lindstrom. Nucleotide sequence of the human nicotinic acetylcholine

receptor beta 2 subunit gene. Nucleic Acids Res. 18(14): 4272. 1990.

Sambrook, J., Fritch, E. F. and T. Maniatis. Molecular Cloning: A Laboratory Manual,

second edition, Cold Spring Harbor Laboratory. Cold Spring Harbor. 1989.

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8. APPENDICES

8.1 GLOSSARY OF TERMS

Genomic Walking

Genomic walking describes the process of sequentially analyzing fragments of a

chromosome starting from a known sequence.

Initiating Primer

A PCR primer for a sequence of interest, which when used in conjunction with

Vectorettes allows the amplification of a DNA fragment adjacent to this sequence.

It is called the initiating primer because it is needed to initiate DNA synthesis in

Vectorette PCR.

Nested PCR

A nested PCR is a second round of PCR using primers internal to the primers used

in the first round of PCR. It allows increased specificity and may produce cleaner

bands.

One-Sided PCR

Any method that enables PCR of a DNA fragment, where only one PCR primer can

be designed to that target sequence. This allows PCR of an uncharacterized

fragment of DNA upstream or downstream of the known region.

PCR

PCR (Polymerase chain reaction) is a method for specifically amplifying fragments

of DNA using two oligonucleotide primers.

Phosphorylated

With reference to oligonucleotides, phosphorylated means that a phosphate group

is attached to 5' end. In normal oligonucleotide synthesis, the oligo is synthesised

unphosphorylated.

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Vectorette

A Vectorette unit is a partially double-stranded fragment of DNA. When ligated to

a suitably cut fragment of target DNA it acts as a template for a Vectorette PCR

primer providing it has been replicated already by DNA synthesis from another

(target) primer. These are sometimes referred to as Vectorette units or

Vectorette ends.

Vectorette I

Vectorette I is the original Vectorette design. It has been used widely in a number

of different applications.

Vectorette II

Vectorette II has the same design as Vectorette I, but contains completely

different sequences. Vectorette II can be used in exactly the same manner as

Vectorette I. In addition it contains the sites for three restriction enzymes (Bam

HI, Eco RI, Hind III) which enables easier sub-cloning of PCR products.

Vectorette Library

A Vectorette library is similar to a cloned DNA library. It consists of a collection of

all possible DNA fragments from a target population, which have been ligated to

Vectorettes.

Vectorette PCR Primer

This is a primer with the same sequence as the bottom strand of the Vectorette.

It can only prime DNA synthesis if this bottom strand has been used as a template

in the first round of PCR.

Vectorette Sequencing Primer

The Vectorette sequencing primer is identical to a portion of the bottom strand of

the Vectorette unit. It can be used to sequence the bottom strand of a Vectorette

PCR product starting from the Vectorette terminal end.

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YAC

Yeast artificial chromosome (YAC). YACs are cloning vectors which allow large

fragments (up to several hundred Kb) to be cloned.

8.2 BUFFERS

Stock Solutions

1 M Tris (1L) Dissolve 121.1 g of Tris base in 800 ml of water. Adjust the pH to

the desired value by adding concentrated HCl.

Desired pH Amount of concentrated HCl

7.4 70 ml

7.6 60 ml

8.0 42 ml

Allow the solution to cool to room temperature before making the final

adjustments to the pH.

Make up the volume of the solution to one litre. Dispense into aliquots and

sterilise by autoclaving.

0.5 M EDTA [pH 8.0] (1L) Add 186.1 g of EDTA.Na2.2H2O to 800 ml of water.

Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (approx. 20

g of NAOH pellets). Make up the volume to 1L and dispense into aliquots.

Sterilise by autoclaving.

1 M MgCl2 (1L) Dissolve 203.3 g of MgCl2.6H2O in 800 ml of H2O. Adjust the

volume to 1 litre. Dispense into aliquots and sterilise by autoclaving.

100 mM ATP ( 1 ml) Dissolve 60 mg of ATP in 800 µl of sterile distilled water.

Adjust the pH to 7.0 with 0.1 M NaOH. Adjust the volume to 1 ml and store in

small aliquots at -20°C.

1 M DTT (20 ml) Dissolve 3.09 g of DTT in 20 ml of 0.01 M sodium acetate (pH

5.2). Sterilise by filtration. Dispense into 1 ml aliquots and store at -20°C.

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TE (1L)

10 mM Tris-Cl[pH 7.6]

1 mM EDTA [pH 8.0]

Combine 10 ml of stock 1M Tris-Cl [pH 7.6] and 2 ml of stock 0.5 M EDTA and

make up the volume to 1 litre with water. Sterilise by autoclaving.

10 x Ligation Buffer (10 ml)

0.5 M Tris-Cl [pH 7.6]

100 mM MgCl2

10 mM DTT

Combine 5 ml of stock 1 M Tris-Cl [pH 7.61], 1 ml of stock 1M MgCl2, 100 µl of 1

M DTT and make up the volume to 1 ml in water. Dispense into aliquots and store

at -20°C.

10 x Lambda Exonuclease Buffer (10 ml)

67 mM Glycine-NaOH [pH 9.41]

2.5 mM MgCl2

Weigh out 503 mg of glycine. Make up the volume to 5 ml with sterile distilled

water and then adjust the pH to 9.4 by adding 1 M NaOH in drops. Add 25 µl of 1

M MgCl2 and make up the volume to 10 ml with sterile distilled water. Dispense

into aliquots and store at -20°C.

TE Equilibrated phenol As required, phenol is removed from the freezer,

allowed to warm to room temperature and melted at 68°C. 8-Hydroxyquinoline is

then added to a final concentration of 0.1 %. The melted phenol is then

extracted several times with an equal volume of buffer (usually 1.0 M Tris pH 8.0,

followed by 0.1 M Tris (pH 8.0) and 0.2 % ß-Mercaptoethanol until the pH of the

aqueous phase is greater than 7.6). The phenol can be stored at 4°C under

equilibration buffer for periods of up to one month.

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