16 Recombinant DNA and Biotechnology 16 Cleaving and

16 Recombinant DNA and Biotechnology

16 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.

16 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.

16 Process of DNA Typing FORENSICS 1. Extract DNA from cell nuclei 2. Restriction Enzymes ------------------> 3. Copy (“amplify”) DNA using PCR 4. 5. Count the STRs at 13 locations … using a Genetic Analyzer – one number comes from each parent – e. g. v. WA 7, 10 Evaluation of Electropherogram

Figure 16. 1 Bacteria Fight Invading Viruses with Restriction Enzymes

16 Cleaving and Rejoining DNA • The enzyme Eco. RI cuts DNA with the following paired sequence: § 5¢. . . GAATTC. . . 3¢ § 3¢. . . CTTAAG. . . 5¢

16 Cleaving and Rejoining DNA • Using Eco. RI on a long stretch of DNA would create fragments with an average length of 4, 098 bases. • Using Eco. RI 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)

16 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 singlestranded 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

16 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