Technological Solutions. In 1977 Sanger et al. were able to work out the complete nucleotide...
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Transcript of Technological Solutions. In 1977 Sanger et al. were able to work out the complete nucleotide...
• 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
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 at each end of the restriction 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 by joining DNA from 2 Different sources
DNA 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 Target 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 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)
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.
• 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 analysis
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.