INTRODUCTION

Most attempts to identify and isolate a novel cDNA result in the acquisition of clones that represent only a part of the mRNA's complete sequence. The missing sequence (cDNA ends) can be cloned by PCR, using a technique called rapid amplification of cDNA ends (RACE). RACE cloning is advantageous for several reasons.

In RACE technique, PCR is used to amplify partial cDNAs representing the region between a single point in a mRNA transcript and its 3' or 5' end. A short internal stretch of sequence must already be known from the mRNA of interest. From this sequence, gene-specific primers are chosen that are oriented in the direction of the missing sequence. Extension of the partial cDNAs from the unknown end of the message back to the known region is achieved using primers that anneal to the preexisting poly(A) tail (3'-end) or to an appended homopolymer tail (5'-end). Using RACE, enrichments on the order of 10 6-to 10 7-fold can be obtained.

3'-RACE (Rapid Amplification of 3'-cDNA Ends)

To generate "3'-end" partial cDNA clones, mRNA is reverse-transcribed using an Anchor Primer that consists of 17 nucleotides of oligo(dT) followed by a unique 20-22 base oligonucleotide sequence. Amplification is then performed using a primer containing part of this sequence that now binds to each cDNA at its 3'-end, and using a primer derived from the gene of interest. A second set of amplification cycles is then carried out using "nested" primers .

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3'- RACE RNA can be analyzed by cloning and sequencing after target region was amplified through RT-PCR. However, there are many difficulties in obtaining full-length cDNA clones from mRNA. RACE (Rapid-Amplification of cDNA Ends) methods, which is performed for cloning the 3′ end or 5′ end region based on the sequence information of cDNA region already obtained, is effective to overcome these difficulties. 3′- RACE is a core set designed to specifically amplify the region which contains 3′-end of target mRNA utilizing 3′-RACE procedure. The supplied Oligo dT-3 sites Adaptor Primer is specially designed for efficient cDNA synthesis from 3′-end of poly(A)+ RNA. Also as the supplied 3 sites Adaptor Primer contains restriction sites of BamH I, Kpn I and Xba I within its sequence, cloning after RT-PCR can be easily achieved. In this case, it is recommended to add the sequence (which is shown as below) at the 5′-side of the upstream specific primer.

1. AMV Reverse Transcriptase XL* (5 units/µl) 20 µl

2. RNase Inhibitor (40 units/µl) 10 µl

3. Oligo dT-3 sites Adaptor Primer** (2.5 µM) 20 µl

4. RNase Free dH2O 500 µl

5. 3 sites Adaptor Primer** (20 µM) 20 µl

6. 10× RNA PCR Buffer(100 mM Tris-HCl (pH8.3), 500 mM KCl)

40 µl

7. dNTP Mixture (ea. 10 mM) 40 µl

8. MgCl2 (25 mM) 80 µl

9. Control F-1-3 sites Adaptor Primer** (20 µM)(upstream primer for positive control RNA)

10 µl

10. Positive control RNA*** (2× 105 copies/µl)(transcribed poly(A)+ RNA of pSPTet3 plasmid)

10 µl

Primer Sequence

Oligo dT-3 sites Adaptor Primer:This primer was designed to have dT region and the restriction sites of BamH I,

Kpn I and Xba I. 3 sites Adaptor Primer:

5′-CTGATCTAGAGGTACCGGATCC-3′ Control F-1-3 sites Adaptor Primer:

5′-CTGATCTAGAGGTACCGGATCCATATCGCCGACATCACCGATG-3′

*** Positive control RNA

Supplied control RNA is in vitro transcribed RNA using SP6 RNA polymerase from plasmid pSPTet3 inserted with DNA fragment (approximately 1.4 kbp) having tetracycline resistant gene, originated from pBR322, in the downstream of SP6 promoter. This control RNA is a poly(A)+ RNA containing 30 bases of poly(A) at the tail. When full-length doublestranded cDNA is synthesized from this control RNA, tetracycline resistant plasmid is obtained by inserting this cDNA.

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Amplified DNA fragments using control RNA and control primers

Principle

Principle

(1) Synthesize 1st strand cDNA by reverse transcription of target mRNA with Oligo dT-3 sites Adaptor Primer

(2) Perform PCR with a gene-specific primer (not supplied) and 3 sites Adaptor Primer

(3) Cut PCR products with an appropriate restriction enzyme(Kpn I or Xba I or BamH I)

(4) Clone the obtained DNA fragments into a proper vector and perform DNA sequencing

5'-RACE (Rapid Amplification of 5'-cDNA Ends)

To generate "5'-end" partial cDNA clones, reverse transcription (primer extension) is carried out using a gene-specific primer to generate first-strand products. Then, a Poly(A) tail is appended using terminal deoxynucleotidyltransferase (TdT) and dATP. Amplification is then achieved to form the second strand of cDNA. Finally, a second set of PCR cycles is carried out using nested primers to increase specificity.

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PROCEDURES

First-Strand cDNA Synthesis by Reverse Transcription

1. Do reverse transcription by assembling the following components on ice.

5 x Reverse Transcription Buffer ------------------------ 4 uldNTPs (10 mM of each dNTP) -------------------------1 ul

0.1 M DTT --------------------------------------------- 2 ulRNasin ------------------------------------------------- 0.5 ul

2. Mix the following components together in an another microfuge tube.

3'-Primer (100 ug/ ul) ----------------------------------- 500 ulPoly A+ RNA ------------------------------------------- 1 ug

(or, Total RNA ------------------------------------------ 5 ug)Add DEPC-treated H2O to make a final volume of ----- 13 ul

Heat for 3 minutes at 80oC and cool rapidly on ice.

Spin for 5 seconds in a microfuge.

Add to the reverse transcription components from step 1.

3. Add 1 ul (200 units) of SuperScript II reverse transcriptase, and incubate for 1 hour at 42oC, and 10 minutes at 50oC.

4. Incubate for 15 minutes at 70oC to inactivate reverse transcriptase.

Spin for 5 seconds in a microfuge.

5. Add 1 ul (1-2 units) of RNase H to the tube and incubate for 20 minutes at 37oC to destroy the RNA template.

6. Dilute the reaction mixture to 400 ul with TE and store at 4oC.

(This is 5'-end non-tailed cDNA pool).

Appending a Poly A+-Tail to First-Strand cDNA

The conditions described below result in the addition of 30-400 nucleotides.

7. Remove excess primer using spin columns (ex. MICROCON-100 spin filters). The final volume recovered should not exceed 10 ul. Adjust volume to 10 ul using

distilled H2O.

8. Add the following components.

5 x Tailing Buffer -------------------- 4 ul1.5 mM CoCl2 ---------------------- 1.2 ul of 25 mM CoCl21 mM dATP ------------------------ 4 ulTdT (Terminal transferase) ---------- 10 units

9. Incubate for 5 minutes at 37oC and then 5 minutes at 65oC.

10. Dilute to 500 ul with TE.

(This is 5'-end tailed cDNA pool).

First Round PCR Amplification

11. Mix the following components in a PCR tube.

5'-end tailed cDNA pool from step 10 1 ul

25 pmole 3'-Primer 2.5 ul of 500 pmole 3'-Primer

25 pmole Oligo dT26-28-Primer for 5'-region 2.5 ul of 500 pmole Oligo dT26-28

5 x Taq DNA Polymerase Buffer 10 ul

1.5 mM dATP 1.5 ul of 50 mM dATP

1.5 mM dCTP 1.5 ul of 50 mM dCTP

1.5 mM dGTP 1.5 ul of 50 mM dGTP

1.5 mM dTTP 1.5 ul of 50 mM dTTP

10% DMSO 5 ul of 100% DMSO

Add distilled H2O to make a final volume of 50 ul

12. Heat in a thermal cycler for 5 minutes at 98oC to denature the first-strand products.

Cool to 75oC.

Add 2.5 units of Taq DNA polymerase.

Incubate for 2 minutes at the appropriate annealing temperature (48-52oC).

Extend the cDNAs for 40 minutes at 72oC.

13. Carry out 30 cycles of PCR amplification using a step program, followed by a 15-minute final extension at 72oC.

Cool to room temperature.

Cycles Time Temperature (oC)

30 30 sec 95oC

30 sec 48-52oC

2-3 min 72oC

1 15 min 72oC

Second Round PCR Amplification

14. Dilute 1 ul of the amplification products from the First Round PCR (from step 13) into 20 ul of TE.

15. Mix the following components in a PCR tube.

20 x Diluted First Round PCR products from step 14

1 ul

25 pmole 3'-nested Primer 2.5 ul of 500 pmole 3'-nested Primer

25 pmole Oligo dT26-28-Primer for 5'-region 2.5 ul of 500 pmole Oligo dT26-28

5 x Taq DNA Polymerase Buffer 10 ul

1.5 mM dATP 1.5 ul of 50 mM dATP

1.5 mM dCTP 1.5 ul of 50 mM dCTP

1.5 mM dGTP 1.5 ul of 50 mM dGTP

1.5 mM dTTP 1.5 ul of 50 mM dTTP

10% DMSO 5 ul of 100% DMSO

Add distilled H2O to make a final volume

of50 ul

16. Carry out 30 cycles of PCR amplification using a step program, followed by a 15-minute final extension at 72oC.

Cool to room temperature.

Cycles Time Temperature (oC)

30 30 sec 95oC

30 sec 52-60oC

2-3 min 72oC

1 15 min 72oC

Cloning

17. To clone the cDNA ends directly from the amplification reaction (or after gel purification, which is recommended), ligate an aliquot of the products to plasmid

vector encoding a 1-nucleotide 3'-overhang consisting of a T on both strands. Such vector DNA is available commercially (TA Kit).

TA CloningPCR products amplified with a non-proof reading DNA polymerase such as Taq

characteristically have single Adenine (A) overhangs at the 3’-end of each synthesized strand of DNA. The PCR product can then be ligated into any

linearized vector having complementary single thymidine (T) nucleotides.  Sequencing

5'- RACE DescriptionRNA can be analyzed by cloning and sequencing after the target region is amplified through RT-PCR. However, there are many difficulties in obtaining full-length cDNA clones from mRNA. The RACE (Rapid-Amplification of cDNA Ends) method is effective to overcome these difficulties. 5′-Full RACE is designed to perform the 5′-RACE procedure. This set achieves amplification of an unknown 5′-end region of mRNA utilizing inverse PCR and allows efficient 5′-RACE.

The inverse PCR method involves a series of restriction digests and ligation, resulting in a looped fragment that can be primed for PCR from a single section of known sequence. Then, like other polymerase chain reaction processes, the DNA is amplified by the temperature-sensitive DNA polymerase. A target region with an

internal section of known sequence and unknown flanking regions is identified Genomic DNA is digested into fragments of a few kilobases by a usually low-

moderate frequency (6-8 base) cutting restriction enzyme. Under low DNA concentrations, self-ligation is induced to give a circular DNA

product. PCR is carried out as usual, with primers complementary to sections of the known

internal sequence

1. AMV Reverse Transcriptase XL* (5 units/µl) 10 µl

2. RNase Inhibitor (40 units/µl) 10 µl

3. 10× RT Buffer (containing dNTP Mix) 15 µl

4. RNase Free dH2O 1 ml

5. RNase H (60 units/µl) 10 µl

6. 5× Hybrid RNA Degradation Buffer 150 µl

7. T4 RNA Ligase (40 units/µl) 10 µl

8. 5× RNA (ssDNA) Ligation Buffer 80 µl

9. 40% PEG#6000 200 µl

10. Positive Control RT-Primer (200 pmol/µl)** 10 µl

11. Positive Control 1st Primer Pair (ea. 20 pmol/µl)** 10 µl

12. Positive Control 2nd Primer Pair (ea. 20 pmol/µl)** 10 µl

13. Positive Control RNA (10 ng/µl) 10 µl

Set components

Primer sequence

Positive Control RT-Primer:5′-(P)AAAATGACCCAG-3′

Positive Control 1st Primer Pair:S1: 5′-AGCGCTTGTTTCGGCGTGGGTATGGTG-3′A1: 5′-CTGGCGATGCTGTCGGAATGGACGATA-3′

Positive Control 2nd Primer Pair:S2: 5′-ACCTACTACTGGGCTGCTTCCTAATGC-3′A2: 5′-TAGATTTCATACACGGTGCCTGACTGC-3′

Principle

1)

Synthesize 1st strand cDNA by reverse transcription from target mRNA using 5′-end-phosphorylated RT primer which is specific to the target RNA

(2) Degradation of RNA in DNA-RNA hybrid by treatment with Rnase H

(3) Circularization of single-stranded cDNA or formation of concatemers by RNA Ligase

(4) DNA Amplification by Nested PCR

Inverse polymerase chain reaction (Inverse PCR)

Is a variant of the polymerase chain reaction that is used to amplify DNA with only one known sequence. One limitation of conventional PCR is that it requires

primers complementary to both termini of the target DNA, but this method allows PCR to be carried out even if only one sequence is available from which primers

may be designed.Inverse PCR is especially useful for the determination of insert locations. For

example, various retroviruses and transposons randomly integrate into genomic DNA. To identify the sites where they have entered, the known, "internal" viral or

transposon sequences can be used to design primers that will amplify a small portion of the flanking, "external" genomic DNA. The amplified product can then be sequenced and compared with DNA databases to locate the sequence which

has been disrupted.The inverse PCR method involves a series of restriction digests and ligation,

resulting in a looped fragment that can be primed for PCR from a single section of known sequence. Then, like other polymerase chain reaction processes, the DNA

is amplified by the temperature-sensitive DNA polymerase:

A target region with an internal section of known sequence and unknown flanking regions is identified

Genomic DNA is digested into fragments of a few kilobases by a usually low-moderate frequency (6-8 base) cutting restriction enzyme.

Under low DNA concentrations, self-ligation is induced to give a circular DNA product.

PCR is carried out as usual, with primers complementary to sections of the known internal sequence.*

Finally the sequence is compared with the sequence available in the data base.

IPCR (Inverse Polymerase Chain Reaction) leads to the amplification of previously unknown sequences because the primers that initially face away from each other on the linear template can be made to face each other as in normal PCR following circularization of the template. Further amplification with nested primers ensures the integrity of the final product, which can be sequenced directly.

Genome Walking by Inverse Polymerase Chain Reaction

                                 

            

PROCEDURES

1. Prepare genomic DNA .Genomic DNA needs to be clean enough to be readily digested by restriction enzymes and not to be inhibitory to ligation of the DNA.

2. Dissolve the DNA in TE.

3. Digest 1 ug DNA with a number (5-7 enzymes) of restriction enzymes in separate tubes in a total volume of 10 ul. Digest to completion.

In order to obtain sequence only from the upstream region, the enzymes chosen must cleave within the known sequence.

In practice, it is easier to cleave with five different enzymes in order to ensure that at least one or two of the digests yield fragments that are neither too large to be

amplified (2-3 kb with Taq polymerase) nor too small to be worth the effort in sequencing the fragment.

4. Heat-inactivate the enzyme at 68oC for 10 min if it is heat-labile or alternatively extract with phenol: chloroform and ethanol precipitate with 1/10th vol 3 M Sodium

acetate (pH 5.2) and 2.5 vol ethanol if heat-stable.

5. After precipitation, wash the DNA pellet with 70% Ethanol and resuspend to 10 ul TE.

6. Take 2 ul (0.2 ug) of heat-inactivated or ethanol-precipitated digested DNA and setup a self-ligation reaction by adding the following components.

Restriction Enzyme Digested DNA 0.2 ug/ 2 ul

10 x T4 DNA Ligase Buffer 10 ul

10 mM ATP 10 ul

T4 DNA Ligase (3 U/ ul) 4 ul

Add distilled H2O to make a final

volume of100 ul

. Ligate 16 hours at 14oC.

8. Remove 10 ul from each tube and add directly to a 100 ul PCR reaction containing the internal pair of the nested primers.

Typical PCR reaction conditions are used as follow.

1 cycle

Denaturation

95oC 60 sec

35-40 cycles

Denaturation

95oC 10 sec

Annealing

Tm-5oC

60 sec

Extension

72oC 3 min

1 cycle

Final Extens

ion72oC

5-10 min

Ethanol precipitate the PCR reactions with 1/10th 3 M Sodium acetate (pH 5.2) and 2.5 vol Ethanol.

10. Run each precipitated DNA in a single well of a 1.2% Agarose gel.

Visualize the bands over a long-wavelength UV-transilluminator.

11. Use the total amount of first-round PCR product in a second round PCR reaction with nested primers.

12. After the temperature cycling is complete, ethanol precipitate and run each product on an agarose gel.

13. Purify the amplified DNA products using any kit.

14. Sequence the PCR product from both directions using nested primers.

AFLP-PCR amplification

The procedure of this technique is divided into three steps:

Digestion of total cellular DNA with one or more restriction enzymes and ligation of restriction half-site specific adaptors

to all restriction fragments. Selective amplification of some of these fragments with two

PCR primers that have corresponding adaptor and restriction site specific sequences.

Electrophoretic separation and amplicons on a gel matrix, followed by visualisation of the band pattern.

Digestion and ligation to adaptors

After selection of genome, the information necessary to perform the experiment must be selected.

 Select restriction enzymes. They will be used to perform a complete theoretical DNA digestion of the genome.   

They all will cleave DNA within recognition sequence The recognized sequence is not ambiguous (no degenerated

nucleotides)  They will all yield overhang ends (no blund ends)

Selective nucleotides introduced in the form must match the upper strand of DNA (see the grey segments in the

picture).

As a result of DNA digestion, three types of DNA fragments will be produced:

Fragments cleaved in both ends by the same restriction enzyme (RE1 or RE2)

Fragments cleaved in 5' end by RE1 and by RE2 in 3' end Fragments cleaved in 3' end by RE1 and by RE2 in 5' end

In AFLP-PCR, only type 2 and 3 fragments will be amplified to yield visible bands. This is due to ligation of different adaptors in each site of the fragment which will allow a geometric increase of this kind of bands when PCR amplification is perform. Adaptors will be ligated to DNA fragments as shown in the picture below.

                                                                                                          

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             Ligation of adaptors in AFLP-PCR experiments

         Some nucleotides of the adaptors are defined by the recognition sequence of the restriction enzymes (in pale blue

and pale green). The sequence of adaptors which does not match the endonuclease recognition sequence (in magenta and red) must be designed to avoid recognition of genomic DNA of the species used in the experiment and to prevent

aberrant results.

                                                                                             

The adaptor sequence must  not regenerate the original recognition sequence. To avoid this regeneration they must be used adaptors that produce a base change in the recognition sequence. An example is shown below:

//-------GAATTC------//------TTAA------////-------CTTAAG------//------AATT------//

 EcoRI               MseI

DNA sequence with EcoRI and MseI recognition sequences

//-------G  AATTC------//------T TAA------// //-------CTTAA G------//-----AAT T------//

DNA restriction

NNNNNNA AATTC------//------T TACnnnnnnNnnn TTTAA  G------//------AAT GNNNNNN

NNNNNNAAATTC------//------TTACnnnnnnnnnnnnTTTAAG------//------AATGNNNNNN

Addition of adaptors.  As nucleotides in red are different to the original ones (blue), restriction sites are not reconstructed.

PCR amplification with adaptor specific primers

In AFLP-PCR experiments, the primers must be designed to allow PCR amplification of the fragments cleaved by RE1 in 5' end and RE2 in 3' end, so that they will be complementary to sequence defined by adaptors and sequence recognised by restriction enzymes. The amplification will be performed as shown in the picture:

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AFLP-PCR with primers matching adaptors and recognised restriction enzymes sequences.

AFLP is composed of 3 steps:1-A) Cellular DNA is digested with one or more restriction enzymes.

Typically this involves a combination of two restriction enzymes: a 4 base cutter (MseI) and a 6 base cutter (EcoRI).

1-B) Ligation of linkers (restriction half-site specific adaptors) to all restriction fragments.

2-A) Pre-selective PCR is performed using primers which match the linkers and restriction site specific sequences.

3) Electrophoretic separation and amplicons on a gel matrix, followed by visualisation of the band pattern.

There are many advantages to AFLP when compared to other marker technologies including randomly amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), and microsatellites. AFLP

not only has higher reproducibility, resolution, and sensitivity at the whole genome level compared to other techniques, but it also has the

capability to amplify between 50 and 100 fragments at one time. In addition, no prior sequence information is needed for amplification .As a

result, AFLP has become extremely beneficial in the study of taxa including bacteria, fungi, and plants.

RAPD PCRRAPD stands for Random Amplification of Polymorphic DNA. RAPD reactions are PCR reactions, but they amplify segments of DNA which are essentially unknown (random)

In RAPD analysis, the target sequence(s) to be amplified is unknown. The researcher will design a primer with an arbitrary sequence. In other words, the researcher simply makes up a 10 base pair sequence (or may have a computer randomly generate a 10 bp sequence), then synthesizes the primer, carries out a PCR reaction and runs an agarose gel to see if any DNA segments were amplified in the presence of the arbitrary primer.

In this figure which depicts a RAPD reaction, a large fragment of DNA is used the template in a PCR reaction containing many copies of a single arbitrary primer.

The arrows represent multiple copies of a primer (all primers (arrows) have the same sequence). The direction of the arrow also indicates the

direction in which DNA synthesis will occur. The numbers represent locations on the DNA template to which the

primers anneal. In this example, only 2 RAPD PCR products are formed:

1) Product A is produced by PCR amplification of the DNA sequence which lies in between the primers bound at positions 2 and 5. 2) Product B is produced by PCR amplification of the DNA sequence which lies in

between the primers bound at positions 3 and 6. Note that no PCR product is produced by the primers bound at positions 1 and 4 because these primers are too far apart to allow completion of the

PCR reaction.

Note that no PCR products are produced by the primers bound at positions 4 and 2 or positions 5 and 3 because these primer pairs are not oriented towards each other.

Finding Differences Between Genomes Using RAPD Analysis

Consider the figure above. If another DNA template (genome) was obtained from a different (yet

related) source, there would probably be some differences in the DNA sequence of the two templates.

Suppose there was a change in sequence at primer annealing site #2:

: RAPD Reaction

                               

                                                     

As shown in this figure, the primer is no longer able to anneal to site #2, and thus the PCR product A is not produced. Only product B is produced.

If you were to run the 2 RAPD PCR reactions diagramed above on an agarose gel, this is what you would see:

RAPD markers are decamer (10 nucleotide length) DNA fragments from PCR amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence and which are able to differentiate between genetically distinct individuals, although not necessarily in a reproducible way. It is used to analyse the genetic diversity of an individual by using random primers. Due to problems in experiment reproducibility, many scientific journals do not accept experiments merely based on RAPDs anymore.

Unlike traditional PCR analysis, RAPD does not require any specific knowledge of the DNA sequence of the target organism: the identical 10-mer primers will or will not amplify a segment of DNA, depending on positions that are complementary to the primers' sequence. For example, no fragment is produced if primers annealed too far apart or 3' ends of the primers are not facing each other. Therefore, if a mutation has occurred in the template DNA at the site that was previously complementary to the primer, a PCR product will not be produced, resulting in a different pattern of amplified DNA segments on the gel.

Allele-specific oligonucleotide

An allele-specific oligonucleotide (ASO) is a short piece of synthetic DNA complementary to the sequence of a variable target DNA. It acts as a probe for the presence of the target in a Southern blot assay or, more

commonly, in the simpler Dot blot assay. An ASO is typically an oligonucleotide of 15–21 nucleotide bases in length. It is designed (and

used) in a way that makes it specific for only one version, or allele, of the DNA being tested. The length of the ASO, which strand it is chosen from, and the conditions by which it is bound to (and washed from) the target

DNA all play a role in its specificity. These probes can usually be designed to detect a difference of as little as 1 base in the target's genetic

sequence.

The human disease sickle cell anemia is caused by a genetic mutation in the codon for the sixth amino acid of the blood protein beta-hemoglobin. The

normal DNA sequence G-A-G codes for the amino acid glutamate, while the mutation changes the middle adenine to a thymine, leading to the sequence G-T-G (G-U-G in the mRNA). This altered sequence substitutes a valine into

the final protein, distorting its structure.

To test for the presence of the mutation in a DNA sample, an ASO probe would be synthesized to be complementary to the altered sequence, here labeled as "S". As a control, another ASO would be synthesized for the

normal sequence "A". Each ASO is fully complementary to its target sequence (and will bind strongly), but has a single mismatch against its non-target allele (leading to weaker interaction). The first diagram shows how the "S" probe is fully complementary to the "S" target (top), but is

partially mismatched against the "A" target (bottom).

A segment of the beta-hemoglobin genes in the sample DNA(s) would be amplified by PCR, and the resulting products applied to duplicate support membranes as Dot blots. The sample's DNA strands are separated with alkali, and each ASO probe is applied to a different blot. After hybridization, a washing protocol is used which can discriminate between the fully complementary and the mismatched hybrids. The mismatched ASOs are washed off of the blots, while the matched ASOs (and their labels) remain.

In the second diagram, six samples of amplified DNA have been applied to each of the two blots. Detection of the ASO label that remains after washing allows a direct reading of the genotype of the samples, each with two copies of the beta-hemoglobin gene. Samples 1 and 4 only have the normal "A" allele, while samples 3 and 5 have both the "A" and "S" alleles (and are therefore heterozygous carriers of this recessive mutation). Samples 2 and 6 have only the "S" allele, and would be affected by the disease. The small amount of 'cross hybridization' shown is typical, and is considered in the process of interpreting the final results.