Technological Solutions技术解的方案

1、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

2、 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 m

3、anufacture 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 siterestriction siterestriction site2 key characteristics of endonucleases make them

4、 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 at each end of the restricti

5、on fragment. short sequences (sticky ends) can form base pairs with complementary sequences. eg. can form a base pair with another restriction fragment formed by the action of the same enzyme on a different strand of dna. dna ligase will seal the gap in the new dna molecule creating recombinant dna

6、by joining dna from 2 different sourcesrecombinant dnadna amplification process of generating a large sample of a target dna sequence from a single gene or dna fragment 2 methods bacterial vector polymerase chain reaction (pcr)bacterial vector relies on the action of restriction endonucleases when t

7、arget sample of dna is treated with an endonuclease it is broken into a specific pattern of restriction fragments based on the enzyme specificity. fragments are spliced into bacterial plasmids generating recombinant dna first recombinant created in 1973 by cohen and boyer recombinant plasmid can be

8、returned to a bacterial cell. as cell multiplies it replicates the plasmids. plasmid serves as a cloning vector (a piece of dna that can contain foreign dna)plasmids in bacteriacreating recombinant dnafigure 4.2bacterial plasmidfigure 4.3practical uses of plasmid vectorsfigure 4.3 (1)practical uses

9、of plasmid vectorsfigure 4.3 (2)practical uses of plasmid vectorsfigure 4.3 (3)practical uses of plasmid vectorsfigure 4.3 (4)practical uses of plasmid vectorsviral vectors viruses can be used as an intermediary.figure 4.4practical uses of viral vectorsfigure 4.4 (1)practical uses of viral vectorsfi

10、gure 4.4 (2)practical uses of viral vectorsfigure 4.4 (3)practical uses of viral vectorsfigure 4.4 (4)practical uses of viral vectorsfigure 4.4 (5)practical uses of viral vectorsfigure 4.4 (6)practical uses of viral vectorsfigure 4.4 (7)practical uses of viral vectorscloning a gene in bacteria video

11、polymerase chain reaction practically an automated method of replicating dna that allows researchers to target and amplify a very specific sequence within a dna sample relies on the action of dna polymerase.process of pcr sample dna fragment is placed in a solution along with nucleotides and primers

12、. solution is heated to break h bonds between base pairs , causing double helix to open. solution is cooled, heat resistant dna polymerase is added and replication begins. cycle is repeated to generate large quantities of sequence in a short time for analysispcr reactionsorting dna fragments third b

13、reakthrough that made sangers 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 analyzedprocess 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 in