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Sanger's method or dideoxy sequencing or chain termination all are same procedures which are involved in use of dideoxynucleotides (ddNTP's) along with normal nucleotides (NTP's) present in DNA. Dideoxynucleotides hold one extra hydrogen group at the 3' carbon as a substitute of hydroxyl group (OH). Addition of ddNTPs stops entry of more nucleotide (Speed, 1992). Because it is not possible to form phosphodiester bond among the dideoxynucleotide and next incoming nucleotide as a result of it DNA chain is stopped.
In first step DNA is denatured by heat resulting into single DNA strand. Then annealing of specifically manufactured primer (3/ end is situated next to DNA sequence of interest) with one end of template. This primer is radio labeled for final detection on gel. After attachment of primer to DNA, whole solution is separated in four tubes labeled "G", "A", "T" and "C". After that DNA polymerase is added along with dNTPs in each tube. Add ddGTP, DDATP, ddTTP, and ddCTP in tubes respectively in one hundredth concentration. Then synthesize DNA. ddNTPs will added in DNA template instead of normal nucleotides and stop chain growth. Run each tube separately in different lane. Run the gel and expose it to UV and observe results (Russell, 2002).
Describe the Maxam-Gilbert sequencing method.
This process is also called chemical sequencing. Allan Maxam and Walter Gilbert in 1976-1977 invent DNA sequencing procedure which stands on chemical modification of DNA along with subsequent cleavage at particular bases (Maxam and Gilbert., 1977). Method includes radioactive labeling on one end and refining of the DNA fragment. As a result of chemical treatment splicing occurs at small region of one or two nucleotide out of four. This occurs in each of four reactions (G, A+G, C, and C+T). It will result into series of radio labeled fragments. Run gel after putting all four reactions in side by side lanes. Expose gel to X-rays film for results. It will show series of bands in relation to radiolabel DNA.
Why is the Sanger method better?
Sanger method is simpler, efficient and in this procedure toxic chemicals used in less amounts along with low levels of radioactivity than the method of Maxam and Gilbert.
What are dideoxynucleotides triphosphates (ddNTPs)?
One -OH is absent at position 3'-C and 2'-C of deoxyribose sugar in Dideoxy nucleoside triphosphates (ddNTPs). One hydrogen is present instead of that OH. phosphodiester bond is formed at 5'-C to previous nucleotide in chain, and at 3'-C position with next dNTP. So as a result of addition of a ddNTP in DNA replication reaction will stop the growth of chain.
What is pyrosequencing?
This technique developed by Pål Nyrén along with his student Mostafa Ronaghi at the Royal Institute of Technology in Stockholm in 1996. Pyrosequencing is process of DNA sequencing which is based on the sequencing by synthesis. In this procedure single strand of DNA is sequenced via enzymatic preparation of complementary strand. We detect activity of DNA polymerase by using chemiluminescent enzyme. In this process one base pair is added and detected at a time at each step. Solutions of C, A, T, and G is added on stationary DNA template and removed after reaction. As a result light is generated when specific nucleotide is attached with template. This sequence of light and chemiluminescent signals enable us to determine the sequence of the template (Ronaghi., 1998).
What do you sequence when you perform EST sequencing?
Transcribed cDNA sequence is determined by using expressed sequence tag or EST sequencing. It is used in determination of gene sequencing (Adams et al., 1991).
What does this picture show (to the left?, to the right?)
Picture shows bands of G, C, A, and T on gel. We can make sequence of DNA template from this information by aligning these bands according to sequence of bands observed on gel. Then image shows dyes that indicates different nucleotides and final results in curve or pick form.
How can you get the pictures above? What I mean is which methods you use to visualize the sequences depicted.
This type of image is observed in Sanger methods or chain termination method.
What is cycle sequencing?
This sequencing process initially produced by Fred Sanger. It is now developed into cycle sequencing reactions. In this technique, reaction has fluorescent dyes. An automated DNA sequencing machine is used for analysis of DNA fragments developed during the reaction. Finally we get order of nucleotide sequence. In this technique cycles of DNA Denaturation, annealing and extension takes place.
Is it possible to sequence RNA?
First convert RNA into cDNA via reverse transcription is needed and then we sequence cDNA and sequencing information about RNA.
Describe sequencing by microarray hybridization!
DNA microarray technology is used in DNA sequencing. It involves in thousand of series of DNA microscopic spots oligonucleotides, known as features. Each spot having picomoles of particular DNA sequence called probes or reporters. These probes are used for hybridization of DNA or cDNA called target. This is done in high stringency conditions. This probe can then be quantified and observed by labeling target with fluorophore silver or chemiluminescence for determination of particular sequence in target. In microarray thousands of probes are used so many testes are used in parallel. This technique is used for studying difference in expression levels and single nucleotide polymorphisms in mutant genome.
The basic principle of technique is between two complementary strands of DNA hydrogen bond is present. Bond strength is directly depends upon the base pairs. Only strong bonds remain intact and hybridized after washing. So generation of signal from florescent label target depends on strength of bonds and hybridization. Final signal power depends on quantity of binding between target and probe present in the spot. In this technique relative quantification is measured and identifying features called as position (Pollack., et al 1999).