Elaborate laboratory chain termination

The laboratory chain termination is a process that will stop the DNA synthesis while preserving the integrity of the newly synthesized tail. This enzyme can be used in conjunction with molecular cloning techniques to obtain pure plasmid DNA. The enzymes which are most commonly used for this purpose are Taq I and Pst I, but there are many other enzymes that could be utilized as well. In order to use this type of enzyme, it must attach itself to one end of a single strand and cut off any further progress down the same piece of DNA. Are you looking for elaborate laboratory chain termination Assignment Help? Worry no more! We got you covered!

It is important when choosing an appropriate enzyme for termination purposes that you make sure it matches up with your desired sequence; otherwise you risk causing mutations or errors in your desired product. One example would be using a T enzyme with too few bases in the recognition sequence, which would result in cutting on both sides of the sequence. On the other hand, using an enzyme with too many bases in the recognition sequence will not give you a clean cut and could just cause your plasmid to circularize.

Single-Stranded Vs Double-Strand

Double-stranded DNA is much more stable than single-stranded.  Although both are held together by hydrogen bonding, double stranded forms a much tighter bond and makes it harder to separate the strands in order to obtain information from each strand.  Single-stranded molecules, however, are very susceptible to breaking apart from denaturing conditions.

steps of chain termination

1. Denature your sample by heating it to 94°C for 5 min in order to separate the double strands of DNA into single strands.
2. Reduce the temperature of your solution to 37°C and add all four deoxynucleotide triphosphates (dNTPs) with 10 U each of the appropriate enzyme. This step requires good timing, because if you are too early, the enzyme will not have enough time to attach itself to one strand and stop progression of DNA synthesis.  On the other hand, if you are too late then there won’t be anything for the enzyme to attach itself to and it will just add more nucleotides
3. Incubate the sample at 37°C for 15 min to let the enzyme finish its job and terminate further DNA synthesis in your desired sequence.
4. Run an aliquot of the reaction on a gel and see if you successfully terminated your DNA synthesis in order to confirm that it was successful.

Mapping and Sequencing

“Mapping and sequencing PCR products is the process of determining the sequence of a DNA region amplified by PCR. This technique can be applied to any size fragments that can be amplified by PCR, and has been used to determine the complete nucleotide sequence of viral genomes as well as entire eukaryotic chromosomes 5, 6. ”

Cycle Sequencing (Sanger)

“The Sanger method involves the use of one labeled, degenerate primer to prime both ends of a target DNA region 7.”

One way to do this is by isolating your PCR product and then purifying it through gel electrophoresis, because this will rid the sample of all extra double stranded DNA that would interfere with successful sequencing.  There are kits available commercially to purify your PCR products before sequencing through gel electrophoresis, such as Pure Link’s Quick Gel Extraction Kit.

Once you have successfully purified your PCR product and ran it on a denaturing gel, you will then need to expose the samples to an X-ray imaging machine in order for them to be successful.  The X-ray film is sensitive to the radiations emitted from your samples and therefore if there is a band on your gel corresponding to your sequence of interest, it will appear as a dark band on the film.

“A critical step in the Sanger method is the removal of unincorporated nucleotide analogs, especially dideoxynucleosides (ddNTPs) and all other triphosphates except for the one at which chain termination occurs.  Removal of these residues is achieved by passing the mixture over a chromatography column and eluting with a low pH buffer”. Sanger’s sequencing method is time consuming and requires exponential amounts of PCR product.

Chain-Termination DNA Sequencing (Solexa)

“The next-generation platform, known as the Solexa platform 9 or Illumina Genome Analyzer, uses reversible dye-terminators and a two-base encoding scheme to resolve four separate colors.  Since each color represents a single base, this method is based on the principle of sequencing by synthesis.”

One way to do this is by isolating your PCR product and then purifying it through gel electrophoresis, because this will rid the sample of all extra double stranded DNA that would interfere with successful sequencing.  There are kits available commercially to purify your PCR products before sequencing through gel electrophoresis, such as PureLink’s Quick Gel Extraction Kit.

Once you have successfully purified your PCR product and ran it on a denaturing gel, you will then need to expose the samples to an X-ray imaging machine in order for them to be successful.  The X-ray film is sensitive to the radiations emitted from your samples and therefore if there is a band on your gel corresponding to your sequence of interest, it will appear as a dark band on the film.

“A critical step in the Sanger method is the removal of unincorporated nucleotide analogs, especially dideoxynucleotides (ddNTPs) and all other triphosphates except for the one at which chain termination occurs.  Removal of these residues is achieved by passing the mixture over a chromatography column and eluting with a low pH buffer.

The sequencing process for the next-generation platform is a relatively simple one.  First, a DNA strand is attached to a sequencing chip via covalent bonds between functional groups on the termini of each single strand and caged biotin molecules on the chip surface 10.  Unincorporated dye terminators are washed away from the sample and then the DNA strand is cleaved by an enzyme to release it.  All four bases are added at once along with all four reversible terminators in a single operation, allowing incorporation of each labeled nucleotide to stop after about one minute 11.”

The third method for sequencing is known as solid-phase minisequencing, which is another sequencing-by-synthesis technique.  “In this method, DNA fragments are synthesized directly onto a solid support (a membrane or bead) that permits direct optical detection of the incorporation event12.” This method can be used either for sequencing nucleic acids using mass spectrometry, or to detect single base substitutions or mutations in DNA.

Improvement of Sequencing Methods

One of the main drawbacks of using X-ray imaging machines to detect bands on gels is that they require a huge amount of space and pose safety issues.  This prevents them from being used for cheaper, large scale projects such as: identifying different strains of bacteria or viruses, sequencing new types of crops to produce more yield, and identifying the many thousands of RNAs inside cells.

Along with this, Sanger sequencing is also time consuming and even though they are most accurate when it comes to determining sequence variation in comparison to next-generation machines (they can detect all single nucleotide polymorphisms), next-generation machines are faster and can sequence a massive amount of DNA in a relatively short period of time.  “In this method, DNA fragments are synthesized directly onto a solid support (a membrane or bead) that permits direct optical detection of the incorporation event12.”

Scientists are now trying to develop sequencing kits that can be used by non-specialists in laboratories, which would make large scale projects much simpler and cheaper.  This could enable scientists to sequence cancer genomes using bioinformatics pipelines to determine the mutation rates in cancer genomes so that new drugs can be developed faster.  Another project scientist is working on involves sequencing genomes of different species in order to determine what genetic material they have in common so that we can get a better idea of how life evolved.

chain-terminating synthesis: conventional DNA sequencing

Before scientists discovered how to sequence long strands of DNA using the chain termination method, they were not able to sequence large amounts of DNA at once.  With traditional sequencing, once a base is read and added onto the new strand, the reaction stops immediately because it is time consuming and there’s no need to add multiple bases.

By using the chain termination method, scientists were able to sequence large amounts of DNA at once.  They sequence many different snippets of DNA and then use software to piece them together and identify the pattern that repeats itself along the way.

Uses for Sequencing

instruments to identify base variations (single nucleotide polymorphisms, SNP).   “In this method, DNA fragments are synthesized directly onto a solid support (a membrane or bead) that permits direct optical detection of the incorporation event12.”

Gel electrophoresis; Identifying proteins; Evolutionary studies; Detecting polymorphisms (SNP) and mutations (point mutations);  are some uses of sequencing .

Conclusion

The lab chain termination method is a technique for amplifying and detecting specific DNA sequences. This option should be used when you need to amplify the target sequence in order to detect it, or if amplification cannot be achieved by PCR due to other factors such as primer mismatch or low template concentration. However, there are some downsides that come with this technique.

For example, false positives can occur because of nonspecific priming events during annealing at locations outside the region of interest on the template strand. In addition, this process can take up more time than traditional PCR methods which makes it less favorable for use in high-throughput sequencing applications where rapid results are needed. But don’t worry! We have experts ready to help you!

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