Question

• Write in detail about “DNA sequencing by Sanger's method”. DNA Isolation, DNA Fragmentation, Amplification and Denaturation, Primer addition and DNA elongation and Significance of ddNPT's are to be explained properly.
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Answer 1

Answer:

Hello mate

Explanation:

Sanger sequencing is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. However, the Sanger method remains in wide use, for smaller-scale projects, and for validation of Next-Gen results.

DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine.

Answer 2

☆DNA sequencing by Sanger's method:

•Friedrick Sanger developed the method in which dna fragments were sequenced using automated DNA sequencers. He is also credited for determining amino acids sequences in proteins.

•so method follows like this:

》For sequencing, the total DNA from a cell is isolated and converted into smaller random fragments as a DNA is very long polymer,

☆ DNA Isolation:

it is just the simple method of isolating the dna from other cellular organelles.

☆ DNA fragmentation:

it is to break the isolated dna to cut into small peices or fragments by endonucleases, and examine.

》Now dna fragments are then cloned in suitable host using specialised vectors.

☆DNA amplification:

it is the process of copying the dna strands by enzyme based process. It can processed by host or any laboratory. it also used in PCR.

☆ DNA denaturation:

process of heating the dna molecules to separate into single strands.

》The cloning results into amplification of each piece of DNA fragments so that it is easy to sequence by many amplified fragments.

here the ☆primer addition occurs which starts the copying of DNA from that attaching it to starting or ori point.

☆DNAelongation: 

• DNA polymerase,enzyme which adds DNA nucleotides to the 3′ end of the newly synthesized polynucleotide strand.
• The template strand specifies which of the four DNA nucleotides A,T,G,C is added at each position along the new chain
• And by this the whole sequence are made.

》The fragments are then sequenced by principle of Sanger's method, sequences were arranged based on some specific sites present in them,which required a specific fragment for sequencing and overlapping on it.

☆ fragments when finally copied and joined are finished by the chain termination by mixing Dideoxynucleotide triphosphates (ddNTPs) . They resulting into partially replicating fragments, the ones we had sequenced for genome.

》Alignment of these sequences are practically humanly not possible.

》Therefore,Computer based programme were developed and used.

》this is what the automated dna sequencing,..now the sequences were properly fixed and assigned to each chromosome, comprising a whole new genome.

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》next was the genetic and physical maps on the genome. This was generated using information on polymorphism of restriction endonucleases recognition sites , and some repetitive dna sequences known as microsatellite(these are the ones which we observe in SAT chromosomes during cell cycle).

》these methodology is also used in DNA fingerprinting.

#note: the commonly used host for cloning were generally bacteria and yeast

and the vectors are called as BAC(bacterial artificial chromosomes) and YAC(yeast artificial chromosomes).

hope its useful!