Transgenic eukaryotes

DNA is introduced into a eukaryotic cell by a variety of techniques, such as transformation, injection, viral infection, or bombardment with DNA-coated tungsten particles As we learned in , when exogenously added DNA that is originally from that organism inserts into the genome, it can either replace the resident gene or insert ectopically. If the DNA is a transgene from another species, it inserts ectopically. (Vectors that replicate autonomously in eukaryotic cells are rare; so, in most cases, chromosomal integration is the route followed.)
The possibility of transgenic modification of eukaryotes such as plants and animals (including humans) opens up many new approaches to research because genotypes can be genetically engineered to make them suitable for some specific experiment. (An example in basic research is in the use of reporter genes. Sometimes it is difficult to detect the activity of a particular gene in the tissue where it normally functions. This problem can be circumvented by splicing the promoter of the gene in question to the coding region of a gene, known as a reporter gene, whose product is easily detectable. Wherever and whenever the gene in question is active, the reporter gene will announce that activity in the appropriate tissue.

Furthermore, because plants, animals, and fungi form the basis for a large part of the economy, transgenic “designer” genotypes are finding extensive use in applied research. A particularly exciting application of transgenesis is in human gene therapy—the introduction of a normally functional transgene that can replace or compensate for a resident malfunctioning allele.

Recombinant DNA technology in eukaryotes

The techniques for gene manipulation, cloning, and expression were first developed in bacteria but are now applied routinely in a variety of model eukaryotes. The genomes of eukaryotes are larger and more complex than those of bacteria, so modifications of the techniques are needed to handle the larger amounts of DNA and the array of different cells and life cycles of eukaryotes. For instance, some eukaryotic proteins cannot be easily expressed in large amounts in bacteria, and eukaryotic expression systems need to be employed. A widely used vector–expression system for eukaryotic proteins is insect baculovirus, into which genes are inserted and expressed at high rates in cultured insect cells,. Although eukaryotic genes are cloned and sequenced in bacterial hosts, it is often desirable to introduce such genes back into the original eukaryotic host or into another eukaryote — in other words, to make a transgenic eukaryote.

Parallelized Sequencing

DNA molecules are physically bound to a surface, and sequenced in parallel.Sequencing by synthesis, like dye-termination electrophoretic sequencing, uses a DNA polymerase to determine the base sequence. Reversible terminator methods (used by Illumina and Helicos) use reversible versions of dye-terminators, adding one nucleotide at a time, detect fluorescence at each position in real time, by repeated removal of the blocking group to allow polymerization of another nucleotide. Pyrosequencing (used by 454) also uses DNA polymerization, adding one nucleotide species at a time and detecting and quantifying the number of nucleotides added to a given location through the light emitted by the release of attached pyrophosphates

Sequencing by Hybridization

Sequencing by hybridization is a non-enzymatic method that uses a DNA microarray. A single pool of DNA whose sequence is to be determined is fluorescently labeled and hybridized to an array containing known sequences. Strong hybridization signals from a given spot on the array identifies its sequence in the DNA being sequenced. Mass spectrometry may be used to determine mass differences between DNA fragments produced in chain-termination reactions.
DNA sequencing methods currently under development include labeling the DNA polymerase, reading the sequence as a DNA strand transits through nanopores, and microscopy-based techniques, such as AFM or electron microscopy that are used to identify the positions of individual nucleotides within long DNA fragments (>5,000 bp) by nucleotide labeling with heavier elements (e.g., halogens) for visual detection and recording.
In October 2006, the X Prize Foundation established an initiative to promote the development of full genome sequencing technologies, called the Archon X Prize, intending to award $10 million to "the first Team that can build a device and use it to sequence 100 human genomes within 10 days or less, with an accuracy of no more than one error in every 100,000 bases sequenced, with sequences accurately covering at least 98% of the genome, and at a recurring cost of no more than $10,000 (US) per genome."

High-throughput Sequencing

The high demand for low-cost sequencing has driven the development of high-throughput sequencing technologies that parallelize the sequencing process, producing thousands or millions of sequences at once. High-throughput sequencing technologies are intended to lower the cost of DNA sequencing beyond what is possible with standard dye-terminator methods.

Molecular detection methods are not sensitive enough for single molecule sequencing, so most approaches use an in vitro cloning step to amplify individual DNA molecules. Emulsion PCR isolates individual DNA molecules along with primer-coated beads in aqueous bubbles within an oil phase. Polymerase chain reaction (PCR) then coats each bead with clonal copies of the DNA molecule followed by immobilization for later sequencing. Emulsion PCR is used in the methods by Marguilis et al. (commercialized by 454 Life Sciences), Shendure and Porreca et al. (also known as "polony sequencing") and SOLiD sequencing, (developed by Agencourt, now Applied Biosystems Another method for in vitro clonal amplification is bridge PCR, where fragments are amplified upon primers attached to a solid surface. The single-molecule method developed by Stephen Quake's laboratory (later commercialized by Helicos) skips this amplification step, directly fixing DNA molecules to a surface.

Large-scale Sequencing Strategies

Current methods can directly sequence only relatively short (300-1000 nucleotides long) DNA fragments in a single reaction. The main obstacle to sequencing DNA fragments above this size limit is insufficient power of separation for resolving large DNA fragments that differ in length by only one nucleotide.

Genomic DNA is fragmented into random pieces and cloned as a bacterial library. DNA from individual bacterial clones is sequenced and the sequence is assembled by using overlapping DNA regions.(click to expand)
Large-scale sequencing aims at sequencing very long DNA pieces, such as whole chromosomes. Common approaches consist of cutting (with restriction enzymes) or shearing (with mechanical forces) large DNA fragments into shorter DNA fragments. The fragmented DNA is cloned into a DNA vector, and amplified in Escherichia coli. Short DNA fragments purified from individual bacterial colonies are individually sequenced and assembled electronically into one long, contiguous sequence. This method does not require any pre-existing information about the sequence of the DNA and is referred to as de novo sequencing. Gaps in the assembled sequence may be filled by primer walking. The different strategies have different tradeoffs in speed and accuracy; shotgun methods are often used for sequencing large genomes, but its assembly is complex and difficult, particularly with sequence repeats often causing gaps in genome assembly.

Dye-terminator Sequencing

Dye-terminator sequencing utilizes labelling of the chain terminator ddNTPs, which permits sequencing in a single reaction, rather than four reactions as in the labelled-primer method. In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with fluorescent dyes, each of which with different wavelengths of fluorescence and emission. Owing to its greater expediency and speed, dye-terminator sequencing is now the mainstay in automated sequencing. Its limitations include dye effects due to differences in the incorporation of the dye-labelled chain terminators into the DNA fragment, resulting in unequal peak heights and shapes in the electronic DNA sequence trace chromatogram after capillary electrophoresis (see figure to the right). This problem has been addressed with the use of modified DNA polymerase enzyme systems and dyes that minimize incorporation variability, as well as methods for eliminating "dye blobs". The dye-terminator sequencing method, along with automated high-throughput DNA sequence analyzers, is now being used for the vast majority of sequencing projects