Sequence analysis of viral genomes

The determination of a DNA virus genome sequence provides the ultimate physical description. While there are methods for sequencing RNA molecules, these methods are not applicable to

Fig. 11.6 The famous Kleinschmidt electron micrograph of phage T4 DNA extruded from the capsid. Before this photograph was made, there was controversy about whether the viral genome was a single piece of DNA or multiple pieces — the fragility of large DNA molecules made them difficult to isolate without shearing. Kleinschmidt took purified bacteriophages and very carefully exposed them to low osmotic pressure. Under the proper conditions, viral DNA was gently released from the capsid and visualized in the electron microscope. Note the presence of two ends, showing that the DNA is linear. (Reprinted with the kind permission of the publishers from Kleinschmidt AK, Lang DJ, Jacherts D, Zahn RK. Darstellung und Längenmessungen des Gesamten Desoxyribonucleinsäure-1 haltes von T2-Bakteriophagen. Biochimica et Biophysica Acta 1961;61:857-864.)

determining the sequence of extremely large molecules such as those that are the genomes of RNA viruses. However, this problem is readily overcome in the study of RNA virus genomes because RNA can be conveniently converted to DNA using appropriate oligodeoxyribonucleotide primers and retrovirus reverse transcriptase. Enzymatic details of the conversion of RNA to cDNA and then double-stranded (ds) DNA are outlined in Chapter 19, Part IV.

DNA sequence analysis requires only a few things: (i) pure DNA; (ii) a method for creating a "nested" set of overlapping fragments, all having one end at the same base and each terminating randomly at different bases in the sequence in question; (iii) a method for labeling these nested fragments at the same site; and (iv) a method of separating the fragments with high enough resolution so that each fragment can be separately resolved.

All the necessary requirements are readily met with the repertoire of techniques available to molecular biologists. Thus, pure DNA can be generated by cloning specific fragments (some of the more basic cloning methods are described in Chapter 22, Part V). Labeling the fragments can be accomplished easily by use of one of a number of enzymatic methods to incorporate a

1. Isolate DNA which will be a template for synthesis of labeled nested set of complementary strands.

5'-DNAX-ATACCGATCGTG-DNAY -3'

2. Anneal short primer complementary to region at 3' end and label with 32P, 35S, or fluorescent dye

3. In four separate reactions extend from primer with limiting amount of a single dideoxy-base-triphosphate to generate nested sets of overlapping fragments.

Reaction 1

with limiting amount of dideoxy-CTP

Reaction 2

with limiting amount of dideoxy-ATP

AC-dnay - 5' * AGCAC-dnay - 5' * ATGGCTAGCAC-dnay - 5' *

Reaction 3

with limiting amount of dideoxy-GTP

GCAC-dnay - 5' * GCTAGCAC-dnay - 5' * GGCTAGCAC-dnay - 5' *

Reaction 4

with limiting amount of dideoxy-TTP

TAGCAC-dnay - 5' * TGGCTAGCAC-dnay - 5' * TATGGCTAGCAC-dnay - 5'*

GATC GATCGATC

--

- -

——

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— —= =

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_ = =

-

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—zz

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1 II 1

Mutant wt

— _

  1. Load products of each reaction onto separate lanes of high-resolution sequencing gel.
  2. Separate fragments by size and read gel from smallest to largest fragment. Sequence will read antiparallel and complementary to the template strand (Why?)
  3. 11.7 Enzymatic sequencing of DNA. The generation of overlapping oligonucleotide sets complementary to a template strand of DNA for sequence analysis was developed by Sanger and colleagues and is described in the text. (a) An outline of the basic method. One major advantage of the method is that it can be used to generate very long sequences with reactions using a single primer site. (b) For example, the gel on the left shows the sequence of a cloned fragment of HSV-1 DNA and the plasmid it is cloned into about 100 bases 3' of the primer site. The sequence can be read as follows:

5'-ACGTC2T2A2GCTAG2C2G2C2TCGC2ATCG2AG5C2TAGT2CGA2TAGCTA-3'

The right gel shows a comparative analysis of the sequence of a wild-type and mutant promoter region for an HSV-1 capsid protein mRNA. This region is about 300 bases 3' of the location of the sequencing primer and shows that high resolution is still readily obtainable as long as the reaction products are fractionated under proper conditions, which in this case are long fractionation times under denaturing conditions. The regions of the two sequences that are different are indicated; the sequences read as follows:

Wild type: 5'-TCACAGGGTTGTCTGGGCCCCTGC-3' Mutant: 5'-TCACAGGACCGGCTGACCGCCTGC-3'

Just above (i.e., 3' of) this region is an example of a typical experimental artifact of this type of sequencing: a spot where there is termination in all reactions due to a structural feature of the sequence in question. Note that the sequence again can be read accurately beyond this point.

nucleotide labeled with a radioisotope (usually phosphorus 32 [32P]or sulfur 35 [35S]) or a fluorescent-tagged nucleotide derivative. Separation of deoxyribonucleotides under denaturing conditions on thin acrylamide gels by high-voltage electrophoresis is sufficiently precise to resolve fragments differing in length by a single nucleotide. More recently, the technique of capillary electrophoresis using a polymer instead of a gel and very small sample sizes has provided high enough resolution to allow the separation of fragments ranging from c. 10 to greater than 1000 bases.

Chemical methods for cleaving DNA at specific bases were originally described by Russian biochemists and perfected for use in DNA sequence analysis by Alan Maxam and Walter Gilbert. Chemical sequencing methods are somewhat laborious, and involve the use of toxic chemicals. They have some advantages, however, and are used for a number of specific applications, most notably at this time for determining the sequence and location vis-à-vis a defined restriction site or point on DNA, which interact with specific DNA-binding proteins.

While chemical sequencing of DNA offers some particular technical advantages and is still occasionally used, enzymatic methods for sequencing are more convenient and are the most frequently used approaches. These methods take advantage of the fact that DNA poly-merase will generate a complementary copy of DNA onto a primer annealed to the template strand.

If a small amount of a dideoxynucleoside triphosphate (which causes chain termination due to lack of a 3'-OH group) is added to the primed synthesis reaction (where the deoxynucleoside triphosphates are in excess), the synthesis of the new DNA strand will terminate wherever the dideoxynucleotide is incorporated. The fact that strand synthesis can only proceed from the primer provides a convenient method for generating overlapping, nested sets of oligonucleotides complementary to any DNA sequence 5' of the primer in question.

The enzymatic method was originally perfected by Sanger and collaborators, and has been modified in many ways. For example and as described a bit later, the method has been automated so that analysis can be carried out and directly entered into computer databases with little human interfacing. The rapid progress made by the human genome project, as well as the increasingly frequent publication of sequences of the entire genomes of free-living organisms, is due to the ease and speed of enzymatic methods. Indeed, where it took several years to determine the complete sequence of HSV-1 (152,000 base pairs) a decade ago, the same problem can now be solved in days! Complete sequence analysis of any virus of interest can be carried out essentially as soon as the virus is isolated and the genome purified.

To generate overlapping oligonucleotides with the same 5' end, all that is needed is a primer sequence that will anneal to a region that is located 3' to the sequence of interest. This is often a region in the vector used to clone the DNA in the first place. Annealing of the primer, which can either be labeled with a radioactive or fluorescent marker, or unlabeled, is followed by enzymatic synthesis of the complementary strand of the DNA template in the presence of a labeled base or bases. After synthesis is allowed to proceed for a short time to ensure the formation of highly labeled material, the reaction is broken into four aliquots and a small amount of a single di-deoxy-base-triphosphate is added to generate oligonucleotides with random stops at a given base. This is shown below for T (remember, lowercase nucleotides signify the complementary base on the antiparallel strand, and DNAY is the region of DNA to which the labeled primer, dnay*, binds):

5-DNAX-ATACCGATCGTG-DNAY-3' tagcac-dnay*—5'

5-DNAX-ATACCGATCGTG-DNAY-3' tggctagcac-dnay*—5'

5-DNAX-ATACCGATCGTG-DNAY-3' tatggctagcac-dnay*—5'

Once generated, the oligonucleotides can then be fractionated on denaturing sequence gels as shown in Fig. 11.7, a schematic representation and examples of actual experimental data. While the separation method is essentially the same as for the chemical method, much less DNA can be loaded, as the labeling can be tailored to the fragment size range to be resolved. This allows higher resolution of the gels.

Automated sequencing takes advantage of the fact that laser light of a given wavelength can excite specific dye molecules to fluoresce at specific frequencies. Different dye molecules fluorescing at different wavelengths can be chemically linked to each of the four di-deoxy-base-tri-phosphates in the reaction mixes described above. These can be used all together in the poly-merase reaction to generate nested products terminating at every base in the sequence. This mixture is then loaded onto a capillary electrophoresis apparatus and subjected to a high voltage. The shortest fragments will, of course, migrate most rapidly through the capillary and past a laser-activated detector where the presence of the terminating, dye-containing fragment will fluoresce at a wavelength characteristic of the terminating deoxynuceleotide. A computer is used to record the order of appearance of the various colored signal peaks. An example of this methodology is shown in Fig. 11.8.

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