PCR amplifies the Eno1gene;given below is the α-enolase protein sequence. This gene that encodes α-enolase is used to design the oligonucleotide PCR primer for sequence amplify cation .There will betwo primers: a forward primer (5′-ATGTCA ATT ATT ACT GAT GTT TAC GCT CGC-3′) that starts from base 1and ends at base30, and a reverse primer (5′-TTATTTTTTCAAGTTGTAGAATGATTTAAGACCACG-3′), that begin at base1263and ends at base1296of the sequence, including the termination cordon.
1-2-1 DNA sequencing
Techniques for sequencing were developed in 1977 by two groups; Alan Maxam and Walter Gilbert, and Fredrick Sanger. Sanger’s method is most commonly used. This method involves the synthesis of a new strand of DNA using the DNA to be sequenced as a template. The reaction begins when single strands of template DNA are mixed with primer, DNA polymerase, the four deoxynucleosides triphosphates (dNTPs), and dideoxynucleoside triphosphates (ddNTPs).In such a reaction mixture, DNA synthesis will proceed until a ddNTP, rather than a dNTP, is added to the growing chain. In order to obtain sequence information, four separate synthesis reactions must be prepared, one for each ddNTP. When each DNA synthesis reaction is stopped,a collection of DNA fragments of varying lengths have been generated. If the DNA is to be manually sequenced, radioactive dNTPs are used and each reaction is electrophoresed in a separate lane on polyacrylamide gel. There are both pros and cons of DNA sequencing. DNA sequencing does lead to invasion of someone’s privacy to some extent. It can be harmful for someone’s medical record and hence proper legal measures need to be followed while DNA sequencing. This technique can be used by doctors to find the medical record’s of patient. During screening doctor can also find a defective gene which might be the cause of disease in the near future and hence correct the gene.