Sequencing DNA means determining the order of the four chemical building blocks - called "bases" - that make up the DNA molecule. The sequence tells scientists the kind of genetic information that is carried in a particular DNA segment. For example, scientists can use sequence information to determine which stretches of DNA contain genes and which stretches carry regulatory instructions, turning genes on or off. In addition, and importantly, sequence data can highlight changes in a gene that may
Sequencing DNA means determining the order of the four chemical building blocks - called "bases" - that make up the DNA molecule. The sequence tells scientists the kind of genetic information that is carried in a particular DNA segment. For example, scientists can use sequence information to determine which stretches of DNA contain genes and which stretches carry regulatory instructions, turning genes on or off. In addition, and importantly, sequence data can highlight changes in a gene that may
Sequencing DNA means determining the order of the four chemical building blocks - called "bases" - that make up the DNA molecule. The sequence tells scientists the kind of genetic information that is carried in a particular DNA segment. For example, scientists can use sequence information to determine which stretches of DNA contain genes and which stretches carry regulatory instructions, turning genes on or off. In addition, and importantly, sequence data can highlight changes in a gene that may
Sequencing DNA means determining the order of the four chemical building blocks - called "bases" - that make up the DNA molecule. The sequence tells scientists the kind of genetic information that is carried in a particular DNA segment. For example, scientists can use sequence information to determine which stretches of DNA contain genes and which stretches carry regulatory instructions, turning genes on or off. In addition, and importantly, sequence data can highlight changes in a gene that may
determining the order of the nucleotides bases adenine, guanine, cytosine and thymine in a molecule of DNA. Maxam-Gilbert sequencing • A sequencing method based on a chemical degradation was described by Maxam & Gilbert (1977). • In this method, end-labelled DNA fragments are subjected to random cleavage at adenine, cytosine, guanine, or thymine positions using specific chemical agents (Table 2). • The chemical attack is based on three steps : • Base modification, • Removal of the modified base from its sugar, • And DNA strand breaking at that sugar positions. • The products of these four reactions are then separated using polyacrylamide gel electrophoresis. • The sequence can be easily read from the four parallel lanes in the sequencing gel. • The template used in this sequencing method can be either double-stranded (ds)DNA or ssDNA from chromosomal DNA. • In general, the fragments are first digested with an appropriate restriction enzyme. Maxam-Gilbert Sequencing
DMS FA H H+S
G G C C G A T C G G T G G C C C A T G C A C A T
Maxam-Gilbert sequencing is performed by chain
breakage at specific nucleotides. Maxam-Gilbert Sequencing
3′ A A G G G+A T+C C C Longer fragments A A A C G T G Shortest fragments C G A G 5′
Sequencing gels are read from bottom to top (5′ to 3′).
Sanger’s sequencing DNA sequencing by the Sanger method
The standard DNA sequencing technique is the Sanger method,
named for its developer, Frederick Sanger, who shared the 1980 Nobel Prize in Chemistry. This method begins with the use of special enzymes to synthesize fragments of DNA that terminate when a selected base appears in the stretch of DNA being sequenced. These fragments are then sorted according to size by placing them in a slab of polymeric gel and applying an electric field -- a technique called electrophoresis. Because of DNA's negative charge, the fragments move across the gel toward the positive electrode. The shorter the fragment, the faster it moves. Typically, each of the terminating bases within the collection of fragments is tagged with a radioactive probe for identification. Chain Termination (Sanger) Sequencing
• A modified DNA replication reaction. • Growing chains are terminated by dideoxynucleotides. Chain Termination (Sanger) Sequencing The 3′-OH group necessary for formation of the phosphodiester bond is missing in ddNTPs.
Chain terminates at ddG Chain Termination (Sanger) Sequencing
• A sequencing reaction mix includes labeled primer
and template.
Primer
5′OP- -3′ OH TCGACGGGC… Template Template area to be sequenced • Dideoxynucleotides are added separately to each of the four tubes. Chain Termination (Sanger) Sequencing
ddATP + ddA A four dNTPs dAdGdCdTdGdCdCdCdG
ddCTP + dAdGddC C four dNTPs dAdGdCdTdGddC dAdGdCdTdGdCddC dAdGdCdTdGdCdCddC
ddGTP + dAddG G four dNTPs dAdGdCdTddG dAdGdCdTdGdCdCdCddG
T ddTTP + dAdGdCddT four dNTPs dAdGdCdTdGdCdCdCdG Chain Termination (Sanger) Sequencing • With addition of enzyme (DNA polymerase), the primer is extended until a ddNTP is encountered. • The chain will end with the incorporation of the ddNTP. • With the proper dNTP:ddNTP ratio, the chain will terminate throughout the length of the template. • All terminated chains will end in the ddNTP added to that reaction. Chain Termination (Sanger) Sequencing
• The collection of fragments is a sequencing
ladder. • The resulting terminated chains are resolved by electrophoresis. • Fragments from each of the four tubes are placed in four separate gel lanes. Chain Termination (Sanger) Sequencing
3′ G G A T C G Longer fragments T A ddG A A T C Shorter fragments A ddG T G 5′
Sequencing gels are read from bottom to top (5′ to 3′).
Automated procedure for DNA sequencing
A computer read-out of the gel generates a “false color” image
where each color corresponds to a base. Then the intensities are translated into peaks that represent the sequence. Summary • Genetic information is stored in the order or sequence of nucleotides in DNA. • Chain termination sequencing is the standard method for the determination of nucleotide sequence. • Dideoxy-chain termination sequencing has been facilitated by the development of cycle sequencing and the use of fluorescent dye detection. • Alternative methods are used for special applications, such as pyrosequencing (for resequencing and polymorphism detection) or bisulfite sequencing (to analyze methylated DNA). If you want Word/PDF??? Soft copy of Word/PDF = 0.013$ (US dollar) (5Rs Indian rupee) Contact :(I will share ppt via Email/WhatsApp/Telegram Email: gnccmysore@gmail.com Telegram:+919738137533(only for Chat) *Once you done payment, Please intimate me.
