Nanopore detectors admit a single DNA strand through a tiny pore and sequence it as it passes through.
Nanotechnology is based on microscopic machinery that operates at the level of single molecules. Nanopore detectors for DNA contain extremely narrow pores that permit a single strand of DNA to pass through one at a time. As the DNA molecule transits the pore, a detector records its presence and its characteristics. Ultimately this should result in a novel method for sequencing individual DNA molecules one at a time. The advantages of nanopore technology are its high speed and its ability to handle long DNA molecules.
A practical nanopore detector consists of a channel in a membrane that separates two aqueous compartments. When a voltage is applied across the membrane, ions flow through the open channel. Since DNA is negatively charged, the DNA is pulled through the nanopore to the positive side. The DNA molecules enter the pore, and are pulled through in extended conformation, one at a time (Fig. 24.16). During the time the channel is occupied by the DNA, the normal ionic current is reduced. The amount of reduction depends on the base sequence (G > C > T > A), therefore, a computer can measure the current, and decipher the sequence based on the differences.
Initial demonstrations have used alpha-hemolysin from Staphylococcus as the channel and a lipid bilayer as the membrane.The mouth of the alpha-hemolysin channel is about 2.5 nm wide—roughly 10 atomic diameters. Double-stranded DNA can enter the pore mouth, but toward the middle, the channel narrows to less than 2 nm, which prevents dsDNA from going any further. The dsDNA remains stuck until the strands separate, allowing single-stranded DNA to pass through the length of the pore.
At present, nanopore detectors can tell apart two 20 nucleotide DNA strands that differ in a single base. It takes approximately a microsecond per base for DNA to transit the pore. Although single-stranded DNA molecules 1000 bases long have been successfully pulled through nanopores, they move so fast through the pores that it is difficult to detect individual bases. Estimates based on future technical improvements suggest the possibility of chips with 500 pores each reading 1,000 bases/second. This could in theory read a bacterial genome in around a minute and read the entire human genome (3 x 109 bases) in less than two hours.
Sequence tags are merely short regions of known sequence in known locations. They are needed when genomes contain vast amounts of non-coding DNA.
Expressed sequence tags come from transcribed regions of the DNA.
Ultimately, all the DNA sequence fragments of a genome must be correlated with a genetic map showing the location of the genes. However, in higher organisms, genes only comprise a small proportion of the DNA. Thus it is necessary to have a series of genetic markers other than genes themselves. We have already discussed sequence motifs such as RFLPs (Ch. 22) and VNTRs and microsatellites (see Ch. 4). However, even the combination of these motifs is not sufficiently specific to map a large genome. Therefore, the human genome was mapped largely by the use of sequence tagged sites (STSs) including expressed sequence tags (ESTs).
A sequence tagged site is simply a short sequence (usually 100-500 bp) that is unique and can be detected by PCR. It is especially important to avoid the repetitive sequences that are found in large numbers on eukaryotic chromosomes. In fact, identification by PCR requires two short sequences to which the PCR primers hybridize, separated by a known length of DNA. The STS is amplified by PCR to give a DNA fragment of specific length (Fig. 24.17).
An expressed sequence tag (EST) is a special type of STS derived from a region of DNA that is expressed, i.e. transcribed into mRNA. Purified mRNA is used to generate the corresponding cDNA by reverse transcriptase. The cDNA is then amplified expressed sequence tag (EST) A special type of STS derived from a region of DNA that is expressed by transcription into mRNA nanopore detector Detector that allows a single strand of DNA through a molecular pore and records its characteristics as it passes through sequence tagged site (STS) A short sequence (usually 100-500 bp) that is unique within the genome and can be easily detected, usually by PCR
B) DNA goes through nanopore
FIGURE 24.16 Principle of the Nanopore Detector
C) DNA CAUSES ELECTRICAL PULSE
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