Technological Solutions技术解决的方案.pptVIP

PCR Reaction Sorting DNA Fragments Third Breakthrough that made Sanger’s Work possible was the development of Gel Electrophoresis Used to separate Molecules according to their mass and electrical charge. Process allows DNA Fragments to be separated so that they can be analyzed Process Solution containing DNA Fragments is applied at one end of a gel. Electric current then applied which causes end of the gel to become polarized. As DNA is acidic it has a negative charge so the DNA will move towards the positive end. Smaller fragments move more quickly. After a period of time they separate into bands creating a DNA Fingerprint. Process refined to the point that Fragments can be separated if they differ by as little as a single nucleotide. Gel Electrophoresis Gel Electrophoresis DNA Fingerprint DNA Fingerprints Technological Solutions In 1977 Sanger et al. were able to work out the complete nucleotide sequence in a virus (Phage 0X174) This breakthrough allowed researchers to use genome sequencing as a way of better understanding the genetics of living cells. Work of Sanger relied on 3 important Developments Discovery of a way to break the DNA strand at specific sites Development of a process to copy or amplify the DNA strand Improvements in the methods for sorting and analyzing DNA Fragments. Restiction Endonucleases As a means of Defending themselves against infection by foreign DNA most prokaryotes manufacture restriction endonucleases Recognize specific short sequence of Nucleotides (target sequence) on a strand of DNA and cut the strand at a particular point within that sequence. This point is the restriction site Restriction Site Restriction Site 2 Key Characteristics of Endonucleases make them useful Specificity Cuts are specific and predictable. Same enzyme will cut the DNA Strand the same way each time. Producing an identical set of smaller pieces Staggered Cuts Most produce a staggered cut that leaves a few unpaired nucleotides remaining on a single strand