What's New in Molecular Markers

AFLP Genotyping and Fingerprinting

1-Ampilified fragment length polymorphisms (AFLPs) are polymerase chain reaction (PCR)-based marker for rapid screening of genetic diversity. AFLPs methods rapidly generate hundreds of highly replicable markers from DNA of any organisms; thus they allow high-resolution genotyping of fingerprinting quality. the time and cost of AFLPs are superior or equal to those of the other markers (allozymes, Random Ampilified Polymorphic DNA(RAPD), Resteraction Enzymes polymorphisms (RFLP) and microsatellites).The AFLP technique is rapidly becoming the method of choice for estimating genetic diversity in both cultivated and natural/rare populations (Fay and Cox 1997; Hill et al. 1996; Karp et al. 1996; Lu et al. 1996; Paul et al. 1997; Qamaruz-Zaman et al. 1997; Sharma et al. 1996; Travis et al. 1996). Additional characteristics of the AFLP technique are described below.
1. It is relatively fast (samples can be processed on automated thermocyclers and DNA sequencers).
2. The technique assays the entire genome for polymorphic markers.
3. It requires relatively small amounts of genomic DNA. Typically 0.05 - 0.5 g of DNA are required, depending upon the size of the genome.
4. It provides 10-100 times more markers and is thus more sensitive than other fingerprinting techniques (e.g., isozymes, RFLPs, microsatellites; Lu et al., 1996; Sharma et al., 1996).
5. Unlike RAPDs, it is highly reproducible. Analyses performed by different workers or in different labs can be compared or reproduced. The bands (DNA fragments) can be run on an automated sequencer that resolves fragment length to single-base units. In addition since each lane incorporates a set of size standards fragment sizes can be estimated accurately thus facilitating comparison of data across gels.
6. Unlike microsatellites, no taxon-specific primer sets are required. Commercially available primers are available that work for most organisms.

Basic Steps of AFLP Fingerprinting

AFLP for complex genomes involves five steps:
I. Restriction of the genomic DNA. Restriction fragments of the genomic DNA are produced by using two different restriction enzymes: a frequent cutter (the four-base restriction enzyme MseI) and a rare cutter (the six-base restriction enzyme EcoRI). Three types of restriction fragments are generated: ones with EcoRI cuts at both ends, ones with EcoRI cut at one end and MseI cut at the other end, and ones with MseI cuts at both ends.

II. Ligation of oligonucleotide adapters. Double-stranded adapters consist of a core sequence and an enzyme-specific sequence. They are specific for either the EcoRI site or the MseI site. Restriction and ligation take place in a single reaction. Ligation of the adapter to the restricted DNA alters the restriction site so as to prevent a second restriction from taking place after ligation has occurred.

III. Preselective amplification. Primers used in this step consist of a core sequence, an enzyme specific sequence and a selective single-base extension at the 3’-end. The sequences of the adapters and restriction sites serve as primer binding sites for the “preselective PCR amplification.” Each preselective primer has a “selective” nucleotide that will recognize the subset of restriction fragments having the matching nucleotide downstream from the restriction site.
The primary products of the preselective PCR are those fragments having one MseI cut and one EcoRI cut, and also having the matching internal nucleotide. The preselective amplification step achieves a 16-fold reduction of the complexity of the fragment mixture.

IV. Selective amplification with labeled primers. Selective primers are either radio-labeled or fluorescently-labeled. They consist of an identical sequence to the preselection primers plus two additional selective nucleotides at the 3’-end (i.e., a total of three selective nucleotides). These two additional nucleotides can be any of the 16 possible combinations of the four nucleotides. From the huge number of fragments generated by the two restriction enzymes, only that subset of fragments having matching nucleotides at all three positions will be amplified at this stage (50-200 fragments). This step reduces the complexity of the PCR product mixture by 256 fold. Different primer combinations will generate different sets of fragments. Preliminary screening is used to choose primer pairs that generate suitable levels of variation for the taxa being studied.

V. Gel-based analysis of the amplified fragments. Because the fragments are labeled with fluorescent dyes, they can be separated and quantified using the Perkin-Elmer/Applied Biosystems Inc. automated sequencer. The GeneScan software analyzes four different fluorescent labels visualized as blue, green, yellow and red. Multiple samples (amplified with separate primer sets, each labeled with a different fluorescent dye) can be loaded in a single gel lane along with an internal DNA size standard (also labeled). Such “multiplexing” reduces the cost of the analysis.

The GeneScan results are displayed as a reconstructed gel image, electropherograms, or tabular data. GeneScan results can be imported into the Genotyper program for subsequent data analysis. This software identifies and measures bands ranging in size from 50 to 500 base pairs. The bands (alleles) are scored as present/absent, and a binary matrix is constructed. The matrix is then analyzed using phenetic methods such as UPGMA and cluster analysis.

AFLP References

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DNA sequencing

INTRODUCTION:
The literature on DNA sequencing is vast with hundred of tricks that are supposed to make the procedure faster and easier. It is impossible to list them all here. The dideoxy terminator method of Sanger and Barrell is the basis for most of the techniques in use today, but there are many different ways of proceeding. For a person who has only modest sequencing needs, it may be wise to let a central facility such as the one here at UNC do the DNA sequencing for you on an automated fluorescent sequencer. Provided you supply a good quality template DNA, you should be assured success with no worry about pouring gels, loading, reading and typing data into the computer. However it is still wise to be able to run your own gels since some types of projects e.g. short sequencing runs to determine if a particular mutant has been made, are probably more efficiently carried out by manual methods.

The major steps in determining a DNA sequence are:

1. Preparation of the template DNA.
2. Annealing of the primer.
3. Carrying out the sequencing reaction.
4. Separating the products on an acrylamide -urea gel.
5. Reading the sequence.
There are several points to be made about each of these steps.
1. Preparation of the template DNA:

You need to decide if you will carry out single stranded sequencing in M13 vectors or double stranded sequencing in plasmids. Formerly nearly all sequencing was done on single stranded molecules and even today single stranded templates probably give the longest and most accurate sequences. However double stranded templates are somewhat easier to prepare and by using two primers in two separate reactions, one can obtain the sequence from both strands. Because of possible errors in sequencing, it is essential that both strands be sequenced, so this is a real advantage of double stranded vectors. You should note that direct sequencing of PCR products is becoming increasingly popular. This is a literature unto itself, and we will say nothing further about it here.
2. Annealing of the primer:

One can use either standard primers specific for vector sequences flanking the template or internal primers which bind to insert sequences. The advantage of using standard primers is that the same primer preparations and annealing conditions can be used over and over. The disadvantage of standard primers is that one can only read a short distance into the sequence e.g. 300-500 bp, which means that if one is trying to sequence a long insert one needs methods of generating subclones in which different sequences are adjacent to the primers. This can be done either by randomly fragmenting the DNA and recloning the fragments into the vector, or by producing either in vivo or in vitro directed deletions from one end of the insert by a variety of methods. These deleted inserts can be recloned and sequenced effectively allowing one to sequence the entire insert.

A second approach is to synthesize new primers complementary to the insert sequence you have just determined and in this way to "walk" down the sequence. The advantage of this procedure is that no manipulation or recloning of the insert is needed. The disadvantage is that one has to continually make new primers and because one is constantly faced with new primers the annealing conditions usually used may not be optimal. However, because primer synthesis is now so rapid and relatively inexpensive and because computer programs are widely available that aid in choosing good primers, more and more sequences are being determined by primer walking.

Even when a primer has been designed, one has to decide if one is going to label the primers or the DNA which is being synthesized. One advantage of primer labeling is that the sequencing bands are more nearly of the same intensity. Primer end labeling during manual sequencing is usually done using 32P-ATP to label the 5’ end of the primer with polynucleotide kinase. 32P labeling has the advantage that it is a relatively high energy beta emmitter which exposes the film in a short period of time allowing faster acquisition of sequence information. Unfortunately, due to its high energy, 32P beta particles scatter over a relatively large area which causes poorer band resolution than when the DNA is labeled internally with 35S. This resolution problem can be avoided by carrying out end labeling with the more expensive 33P-ATP which has beta particle emissions which are only about one fifth the strength of 32P. This provides less sensitivity than 32P, but provides excellent resolution.

3. Carrying out the sequencing reaction:



The enzyme used historically in the overwhelming number of sequencing reactions is Sequenase which is a modified form of bacteriophage T7 DNA polymerase. This enzyme has many advantages over the Klenow fragment of DNA polymerase I which was formerly used. It is more processive and produces relatively uniform peak heights-especially if Mn++ is used in the reaction buffer. This leads to increased accuracy in sequence determination. In the past, 32P labeled dNTPs were used to label the DNA product. However more recently workers have turned to 35S or 33P labeled dNTPs which provide higher resolution (see the discussion above about annealing of the primer).

More recently, PCR based sequencing methods, called cycle sequencing, have begun to supplant the methods based on Sequenase. The general approach is very similar. However, cycle sequencing has several advantages. First, only minute quantities of template DNA are required and this DNA can often be relatively crude. Secondly, because the sequencing reaction are carried out with a thermostable enzyme at high temperatures, templates with extensive secondary structure do not prevent one from obtaining clear sequencing results. A recent reference to this area is Fan et al. Biotechniques 21:1132-1137, 1996
4. Separating the products on an acrylamide-urea gel:

There have been many advances in gel technology since the original systems developed in the late 1970s. One major improvement was gradient gels which made band spacing more uniform and led to the ability to read an extra 50-100 bases. Initially the gradients were made by incorporating an acrylamide and salt gradient into the gel as it was poured. Alternatively the gel could be varied in thickness by using tapered spacers. The easiest procedure however is to simply add a high concentration of NaOAc to the lower gel reservoir as the gel is running. This creates the necessary gradient with minimal difficulty.

Various improvements in the gels themselves include making gels stronger by the addition of linear acrylamide, or using other gel forming ingredients which give higher resolution (Long Ranger). In addition the process of pouring the gels has been simplified by omitting the need for taping. Some workers have developed ultrathin gels which give higher resolution. Devices have also been invented to collect the separated bands as they emerge from the gel (bottom wipers) which are only now becoming commercially available.

Silver staining is a relatively new procedure for determining DNA sequences which minimizes many of the steps needed to produce autoradiograms and obviates the need to use radioactivity.

5. Reading the sequence:

Automatic film readers are now available which take much of the tedium out of reading sequences. The various types range from models which merely record the sequence as you push a button indicating the base, to units which carry out the entire process automatically. We have little experience with these more advanced models.
Hopefully this very brief discussion will alert you to the many various techniques that exist. We have not even discussed the many radically new sequencing methods that are being developed for the human genome project. Most of these are currently interesting only to the experts, but as some of these systems become commercially