Recombinant DNA and Biotechnology

Cleaving and Rejoining DNA Recombinant DNA technology is the manipulation and combination of DNA molecules from different sources. Recombinant DNA technology uses the techniques of sequencing, rejoining, amplifying, and locating DNA fragments, making use of complementary base pairing.

Cleaving and Rejoining DNA Bacteria defend themselves against invasion by viruses by producing restriction enzymes which catalyze the cleavage of DNA into small fragments. There are many such enzymes, each of which recognizes and cuts a specific sequence of bases.

Process of DNA Typing FORENSICS Extract DNA from cell nuclei Restriction Enzymes -----------------------------------> Copy (“amplify”) DNA using PCR Count the STRs at 13 locations … using a Genetic Analyzer – one number comes from each parent – e.g. vWA 7, 10 Evaluation of Electropherogram

Figure 16.1 Bacteria Fight Invading Viruses with Restriction Enzymes

Cleaving and Rejoining DNA The enzyme EcoRI cuts DNA with the following paired sequence: 5¢ ... GAATTC ... 3¢ 3¢ ... CTTAAG ... 5¢

Cleaving and Rejoining DNA Using EcoRI on a long stretch of DNA would create fragments with an average length of 4,098 bases. Using EcoRI to cut up small viral genomes may result in only a few fragments. For a eukaryotic genome with tens of millions of base pairs, the number of fragments will be very large. Hundreds of restriction enzymes have been purified from various organisms, and these enzymes serve as “knives” for genetic surgery, OR “SCISSORS”.

Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 1)

Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 2)

Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 3)

Cleaving and Rejoining DNA Electrophoresis gives two types of information: Size of the DNA fragments can be determined by comparison to DNA fragments of known size added to the gel as a reference. A specific DNA sequence can be determined by using a complementary labeled single-stranded DNA probe. The specific fragment can be cut out as a lump of gel and removed by diffusion into a small volume of water.

Figure 16.4 Cutting and Splicing DNA

Getting New Genes into Cells Bacteria have been useful as hosts for recombinant DNA. Bacteria are easy to manipulate, and they grow and divide quickly. They have genetic markers that make it easy to select or screen for insertion. They have been intensely studied and much of their molecular biology is known.

Figure 16.5 (b) Vectors for Carrying DNA into Cells