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 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

Recombinant DNA

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)

Plasmids in Bacteria

Creating Recombinant DNA

Figure 4.2

Bacterial Plasmid

Figure 4.3

Practical Uses of Plasmid Vectors

Figure 4.3 (1)

Practical Uses of Plasmid Vectors

Figure 4.3 (2)

Practical Uses of Plasmid Vectors

Figure 4.3 (3)

Practical Uses of Plasmid Vectors

Figure 4.3 (4)

Practical Uses of Plasmid Vectors

Viral Vectors

• Viruses can be used as an intermediary.

Figure 4.4

Practical Uses of Viral Vectors

Figure 4.4 (1)

Practical Uses of Viral Vectors

Figure 4.4 (2)

Practical Uses of Viral Vectors

Figure 4.4 (3)

Practical Uses of Viral Vectors

Figure 4.4 (4)

Practical Uses of Viral Vectors

Figure 4.4 (5)

Practical Uses of Viral Vectors

Figure 4.4 (6)

Practical Uses of Viral Vectors

Figure 4.4 (7)

Practical Uses of Viral Vectors

Cloning a Gene in Bacteria

• Video

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

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