Microarray Method for Genetic Testing

Genetic testing allows the genetic diagnosis of vulnerabilities to inherited diseases, and can also be used to determine a person's ancestry. Normally, every person carries two copies of every gene, one inherited from their mother, one inherited from their father. The human genome is believed to contain around 20,000 - 25,000 genes. In addition to studying chromosomes to the level of individual genes, genetic testing in a broader sense includes biochemical tests for the possible presence of genetic diseases, or mutant forms of genes associated with increased risk of developing genetic disorders. Genetic testing identifies changes in chromosomes, genes, or proteins. Most of the time, testing is used to find changes that are associated with inherited disorders. The results of a genetic test can confirm or rule out a suspected genetic condition or help determine a person's chance developing or passing on a genetic disorder. Several hundred genetic tests are currently in use, and more are being developed. 

DNA Sequencing Method: Cycle Sequencing

To sequence a piece of dna you need 1)a Template DNA 2) a short DNA primer that is complementary to the dna you want to sequence, 3)A enzyme called DNA polymerase,(4) Four nucleotides.(A,C,G,T), To this mix ,we also add a second type of nucleotide; one that has a slightly different chemical formula, These dideoxynucleotides(diddtp) can be recognized by a DNA sequencer.

To start the sequencing reaction this mixture is heated to 96C ,so the template DNA's two complementary strand separates,Then the temperature is lowered, so that the short "primer" sequence finds its complementary sequence in the template DNA.Finally the temperature is raised 60c,this allows the enzyme to bind to the DNA and create a new strand of DNA. 

The sequence of this new DNA is complementary to the original DNA strand. The enzyme makes no distinction between dNTPs or didNTPs.each time a didNTP is incorporated, in this case didATP,The synthesis stops. Because billion of DNA molecules are present in the test tube, the strand can be terminated at any position. This results in collection of DNA strands of many different lengths.

The sequencing reaction is transferred from the test tube to a lane of a polyacrylamide gel. The gel is placed into a DNA sequencer for electrophoresis and analysis. The fragments migrate according to size and each is detected as it passes a laser beam at the bottom of the gel. Each type of dideoxynucleotide emits colored light of a characteristic wavelength and is recorded as a colored band on a simulated gel image, and finally computer program interprets the raw data and outputs an electropherogram with colored peaks representing each letter in the sequence.the sequence fragments are sorted out according to the size, starting from the shortest to longest one, the stimulated gel image is read from bottom to top, starting with the smallest fragment, Thus we sequences present in template DNA.

UniSeq DNA Sequencing System

UniSeq™ is a universal DNA sequencing primer walking technology developed by Nucleics for large scale DNA sequencing applications like whole genome sequencing projects. The UniSeq DNA sequencing library & software offers the following advantages over other sanger DNA sequencing methodologies:

• A fast and cost effective universal primer walking approach
• Very simple and robust protocol
• Compatible with all Sanger DNA sequencing technologies and equipment
• Fully adaptable to high throughput, large scale DNA sequencing facilities

The UniSeq DNA sequencing system

The UniSeq DNA sequencing system utilizes a unique methodology to reliably create template specific DNA sequencing primers. The process of generating each UniSeq DNA sequencing primer involves the addition of an "E"- and "T"-oligonucleotide, together with a specifically formulated additive mixture, directly to the sequencing reaction. These oligonucleotides (termed EO and TO) hybridize during the sequencing reaction to produce the template specific primer (S-primer) (Figure 1).

Figure 1. Generation of template specific UniSeq S-primer. The EO hybridizes to the TO and gets extended to form the S-primer. N: degenerated oligonucleotide positions. X: specific positions of variable nucleotide value.
By selecting specific EO and TO oligonucleotides from a small, pre-synthesised library of 768 oligonucleotides, over 131,000 different, template specific primers can be created. This simple combinatorial effect forms the basis for the high specificity and universal applicability of the UniSeq system in DNA sequencing.
The UniSeq system is fully compatible with the common DNA sequencing reagents (e.g. BigDye™ from Applied Biosystems or DYEnamic™ from Amersham Biosciences) and gives excellent results using modern capillary DNA sequencers (Figure 2).

Figure 2. UniSeq reaction sequencing trace. Two hundred nanograms of plasmid DNA was sequenced with BigDye vers.3.1 and UniSeq primers EO 18 and TO 447. The DNA sequencing reaction was purified by ethanol precipitation and analyzed on an ABI 3700 DNA sequencer.
The UniSelect™ DNA sequencing software is provided for the automated and optimized selection of the optimal EO & TO primer pairs for each DNA template. The UniSelect software also supplies an automated interface to control the robotic pipetting of the UniSeq oligonucleotides, DNA templates and other required sequencing reagents.
To aid the finishing of Whole Genome Sequencing (WGS) projects, an additional software package (UniFinish™) is available. UniFinish is able to parse ACE format assembly files and select the EO/TO oligonucleotides, together with the appropriate DNA templates, required to close non-physical DNA sequence gaps in the genome assembly.

Feature and benefits of UniSeq

Genomes are finished faster

The UniSeq DNA sequencing system provides a competitive alternative to DNA sequencing with custom synthesized oligonucleotides. While many advances have been made in the automation of oligonucleotide synthesis, this process is still complex (eg. requiring high maintenance machinery) and slow (several hours per synthesis). These limitations have become extremely critical for high throughput DNA sequencing facilities where modern capillary DNA sequencers have reduced the separation time to one to two hours. The generation of specific DNA sequencing primers by the UniSeq process during the DNA sequencing reaction step (i.e. no time-costs) breaks the current bottleneck imposed by custom oligonucleotide synthesis and allows for the most efficient utilization of existing DNA sequencing instruments and other equipment.

Lower per finished base costs

Currently, two major strategies are used in WGS – Random Shotgun DNA Sequencing (RSS) and Primer Walking DNA Sequencing (PWS). Most WGS projects currently employ RSS, especially in early stages of projects. However, RSS requires generating a large amount of redundant DNA sequence (commonly 6 to 15 times the genome size) for assembly purposes. The alternative PWS strategy requires relatively little redundant DNA sequence data, however, it requires a large numbers of custom made oligonucleotide primers. The high costs (US$3 to 5 per primer), together with the required synthesis delay, have prevented the general adoption of PWS strategy in WGS Genomics projects.
UniSeq provides the advantages of both WGS strategies. It offers the speed and simplicity of the RSS approach, while providing the data efficiency inherent with the PWS approach. Computer simulations and limited trials have shown that UniSeq DNA sequencing system offers cost and time savings of greater than 80% over current WGS approaches.

Increased DNA sequencing flexibility

Nucleics has extensively tested UniSeq in house in a number of general DNA sequencing and WGS projects. In addition, Nucleics has formulated novel strategies for the easy and smooth implementation of UniSeq into industrial scale DNA sequencing facilities. We also offer consulting services to help integrate UniSeq into new or existing DNA sequencing facilities.

DNA Sequencing: Shotgun Sequencing

Shotgun Sequencing was first developed by Craig Ventor in 1996. He developed it as he was working in the Genome Research Institute. It was him who also made it popular. He then went on to start his Celera Corporation with the sole mission and goal of doing the sequencing of mainly the human genome in as little as three years. But of course, some say that in genetics, the shotgun sequencing was in fact first developed by the double Nobel laureate, Fred Sanger, in the 1970s .
Shotgun Sequencing is a DNA sequencing method that involves the physically breaking down of a long stretch of DNA into very small fragments; about 2,000 base-pair. These fragments are then cloned, sequenced and also assembled with the use of computer analysis.
Also known in other circles and by other people as shotgun cloning, this method is said to be one of the harbinger technologies which is mainly responsible for bringing about what we now have as full genome sequencing. The sequencing of the human genome was done by Craig Ventor’s Celera Corporation as well as by the Human Genome Project. They used a map-based sequencing while Craig’s Ventor’s Celera used shotgun sequencing.
Today, however, Craig Ventor’s Shotgun Sequencing is the preferred system and method for doing other types of genome sequencing. And as science and technology improves, it’s clearly obvious that more things will be discovered that will alter or improve the method, for the better. For example, there is now the method called the next-generation sequencing, which is said to result in high coverage than Craig’s. As they say, only time will tell what the future has for this and other methods yet unravelled because man is discovering newer things each and every day. And as many continue to share what they know with others, better methods will obviously be discovered.

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Separation of DNA Fragments Using PAGE Method

This method is able to separate DNA fragments with the size of as small as 10 bp and up to 1 kb with the resolution of as little as 1 bp. While agarose gel electrophoresis is only able to separate DNA fragments with the bigger size that PAGE does or in the size range of 100 nucleotides to around 10 – 15 kb.





Materials:

• Gel apparatus: Many designs of apparatus are commercially available. The gel is poured between two vertical plates held apart by spacers
  
  
  
  
  
  
  
  
  The plates should be cleaned thoroughly and then wiped with ethanol. To help ensure that the gel only sticks to one plate when the apparatus is disassembled, apply silicon to one of the gel plates. This is easily done by wiping the plate with a tissue soaked in dimethyl dichlorosilane solution and then washing the plate in distilled water followed by ethanol. If the plates are baked at 100^oC for 30 min, the siliconization will last four to five gel runs.

  
  
  
  
  
  
• Deionized H₂O: Autoclaved water is not necessary for the gel mix or running buffer, but it should be used for diluting samples and purification from gel slices.
• l0x TBE: 108 g of Trizma base (Tris), 55 g of boric acid, and 9.3 g of ethylenediaminetetraacetic acid (EDTA) (disodium salt). Make up to I L solution with deionized H₂O, which should be discarded when a precipitate forms.
• Acrylamide stock: 30% acrylamide, 1% N,N'-methylene bisacrylamide. Store at 4^oC. This is available commercially, or it can be made by dissolving acrylamide and bisacrylamide in water, which should be filtered. Acrylamide is a neurotoxin and therefore must be handled carefully. Gloves and a mask must be worn when weighing out.
• APS: 10% Ammonium persulphate (w/v). This can be stored at 4^oC for 1-2 months.
• TEMED: N,N,N',N'-tetramethyl-l,2-diaminoethane. Store at 4^oC.
  
• 5X sample buffer: 15% Ficoll solution, 2.5X TBE, 0.25% (w/v) xylene cyanol and 0.025% (w/v) bromophenol blue.
• Ethidium bromide: A l0-mg/mL solution. Ethidium bromide is a potent mutagen and should be handled with care. Store at 4^oC in the dark.

Methods:

• For 50 mL, enough for a 18 x 14 x 0.15 cm gel, mix l0x TBE, acrylamide, H₂O, and APS as described in Table below.

• Just prior to pouring, add 50 microliters of TEMED and mix by swirling.
• Immediately pour the gel mix between the gel plates and insert the gel comb. Leave to set; this takes about 30 min.
• Fill the gel apparatus with 0.5X TBE and remove the comb. Use a syringe to wash out the wells, this may take multiple washes. It is important to remove as much unpolymerized acrylamide as possible because this impairs the running in of the samples
  
  
  
  
  
  If you are separating very small fragments, e.g., less than 50 bp, the gel should be prerun for 30 min, as this elevates the resolution problem experienced with fragments running close to the electrophoresis front..

  
  
  
  
  
  
  
  
• Add 0.2 volume of 5x sample buffer to each sample, usually in 10-20 microliters of TE, water, or enzyme buffer. Mix and spin the contents to the bottom of the tube
  
  
  
  
  
  High-salt buffers (above 50 mM NaCl) will affect sample mobility and tend to make bands collapse. In this case, salt should be removed by ethanol precipitation..

  
  
  
  
  
  
• Load the samples on the gel and run at 200-300 V (approximately 10 V/cm) until the bromophenol blue band is two-thirds of the way down the gel; this takes about 2.5 h
  
  
  
  
  
  Do not run the gel faster than 10 V/cm, as this will cause the gel to overheat, affecting the resolution. The gel can be run more slowly, e.g., 75 V will run overnight.

  
  
  
  
  
  
  
  
• Disassemble the gel apparatus and place the gel to stain in I mg/mL of ethidium bromide for approximately 30 min. View the stained gel on a transilluminator.

Happy separating DNA fragments.

Purification Of Plasmid DNA

After the initial characterization, it is possible to purify further some or all of the plasmid DNAs by RNase digestion and extraction with organic solvents. This further purified DNA is suitable for techniques such as DNA sequencing, subcloning or the production of gene probes. In order to purify plasmid DNA after the isolation process, any residual RNA and contaminating protein are removed. This purification step involves two main steps, which are, first, removing residual RNA by using RNase in order to digest RNA and, second, extract contaminating protein using organic solvents, phenol-chloroform.


Materials:

• RNase A: Make up as a solution in water at 10 mg/mL, Heat for 10 min in a boiling water bath or heating block to eliminate any DNase activity. Aliquot and store at -20^oC.
• 0.4 M Ammonium acetate.
• Chloroform= A 24: 1 mix of chloroform and isoamyl alcohol. Store at 4^oC.
• Phenol/chloroform= 25:24: 1 mix of TE-equilibrated phenol, chloroform, and isoamyl alcohol. Store at 4^oC.
• 100% Ethanol.
• Sterile wooden toothpicks.

Methods:

• Add 50 microliters of 4 M ammonium acetate containing 200 micrograms/mL RNase A to each miniprep and incubate it at room temperature for 20 min.
• Add 100 microliters of phenol/chloroform to each DNA preparation.
• Vortex briefly and centrifuge at high speed for 2 min in a microfuge. Remove the top layer containing the DNA and place it in a new sterile tube.
• Add 100 microliters of chloroform to each tube.
• Vortex briefly and centrifuge at high speed in a microfuge for 2 min. Remove the DNA in the top layer and place it in a second sterile tube.
  
  
  
  For phenol/chloroform extractions avoid removing material from the interface.
  
  
• Add 200 microliters of 100% ethanol to each tube.
• Shake briefly to precipitate the DNA and centrifuge at high speed for 5 min at room temperature.

Done. Now you can extract, isolate, and purify Plasmid DNA using methods which I had described in this blog. Hopefully those can be useful for you.