The base sequence of DNA fragments that have been cloned can be determined In these methods nucleotide sequence of DNA is to be determined. DNA to Protein Sequence can be find out by Expasy Tool.
To check DNA sequencing appropriate methods are used to generate DNA fragments that end at the four bases adenine, thiamin, guanine, and cytosine. After making fragments these fragments run on capillary Gel Electropherosis and molecule of different molecular length are separated on gel.
Two Different DNA Sequencing Procedure
Maxam Gilbert Sequencing
Maxam- Gilbert labeled DNA fragment with 3P as its 5’ end. And then four nucleotide bases are separated by using a chemical in such a way that each nucleotide brake per chain.
Four separated test tubes for each base is prepared as bases are A, T, G , C. After this step gel is prepared and these labeled bases ran on gel.
After gel electrophoresis and autoradiography only those bases which were labeled with 3P will show up on gel.
This chemical method is only seldom used because this method is time consuming and difficult to handle toxic chemicals.
Sanger method of DNA Sequencing
Sanger full name was Frederick Sanger, he won Nobel Prize in chemistry in 1980. Sanger method is also called dideoxy method. It is most used Biochemistry technique.
Why this method is called dideoxy method?
As dTTP has OH at 3’ end but in case of dideoxy method there is a lack of OH group at 3’ end. ddTTP stand for dideoxythymidinetriphosphate.
This ddTTP can be added in DNA strand for its growth but it could not possible due to lack of OH group at 3’ end and chain elongation method will stop.
Reason of Chain Termination
There is no OH group which attaches next nucleotide for chain elongation. That’s ways ddTTP is also called chain termination method.
Steps Involved in Sanger Sequencing Method
A single strand of DNA sequenced is used as template
Take these four dideoxynucleotide labeled with a “tag” that fluorescence a different color.
Took all for normal neucleotide sample
DNA polymerase used for DNA synthesis
DNA Sequencing Procedure by Sanger Method
DNA polymerase enzyme use dNTPs as a substrates and added in to a primer. DNA polymerase enzyme adds ddNTPAs instead of normal dNTPAs. The primer is hydrogen bonded to 3’ end of the DNA to be sequenced.
DNA template is denatured by heating and then cooled and primer can bind to single stranded template
After primer attached again temperature is raised which allow DNA polymerase enzyme to synthesized new DNA starting from the primer. DNA polymerase adds nucleotide, nucleotide continuous to add until dideoxy add which will end the strand elongation.
At the end of incubation period, the fragment is separated by capillary Gel electrophoresis. In which short fragment move quickly and long will move slow. The resolution is so good that a difference of one nucleotide is enough to separate that strand from next shorter and next longer strand
Each of four dideoxynucleotides fluorescence a different color when illuminated by laser beam and an automatic scanner provides a printout of sequence.
Advantages of Sanger Sequencing
It can be used RNA as well as DNA sequence. If you want to sequence of RNA, a single strand DNA copy is made using RNA as a template by using an enzyme which is called reverstrascriptase. Various sized DNA fragment are generated for Sanger type sequence in the presence of ddNTPAs by making single strand DNA.
Cost of DNA sequencing
According to CNBC, It shows the cost to sequence a genome diverging drastically around 2008, falling from almost $10 million to close to $1,000 today.
The first human genome took $2.7 billion and almost 15 years to complete. Now, according to Cowen analyst Doug Schenkel, genome sequencing and analysis cost around $1,400
DNA Sequencing Services
DNA sequence services provided by molecular biology labs in USA. North America provide high quality services by trained staff.
GENEWIZ also provide services of Sanger sequencing within 24 hours.
Speedy sequence DNA sequencing provide costumer services of DNA sequencing in different costs. Speedyseq DNA sequencing in 1 reaction price is $7.50. PCR clean – up for Sanger sequencing in 1 sample is $5.00.
Next Generation Sequencing
NGS has r evolutionised genomic research. By using NGS one can sequenced human genome in a single day. This is also known as high-throughput sequencing, is the term which is used to describe number of different modern technique
Roche 454 sequencing
Iron torrent; proton / PGM sequencing