Biotechnology Methods

1. Biotechnology Methods Producing Recombinant DNA Locating Specific Genes Studying DNA Sequences

2. Recombinant DNA • DNA Produced by Joining Segments of DNA From Different Sources • eg. Bacterial plasmid DNA + Human DNA to Produce Human Insulin

3. Tools for Producing Recombinant DNA Restriction enzymes: enzymes that cleave the DNA double helix at specific nucleotide sequences

4. Results in sticky end sticky end Use of the Restriction Enzyme Bam H1 5’— G G A T C C — 3’ 3’— C C T A G G — 5’ 5’— G G A T C C — 3’ 3’— C C T A G G — 5’ DNA fragments from different sources can be joined together if they have complementary sticky ends.

5. Producing Recombinant DNA

6. Tools for Producing Recombinant DNA Plasmid: extrachromosomal, independently replicating, small circular DNA molecule; a type of vector used to carry DNA into bacterial cells

7. ampR Bam H1 tetR ampR If additional DNA is inserted at the Bam H1 site, the tetR gene is disrupted and is no longer functional DNA Inserted in a Plasmid Can be Detected by Disruption of a Selectable Marker

8. restriction enzyme restriction enzyme Producing Recombinant DNA Mix together Add DNA Ligase Many recombinant DNA molecules are produced, each with a different piece of source DNA Treat source DNA with restrictionenzyme Treat plasmid DNA with same enzyme Transform bacterial cells Each bacterial cellcarries a different recombinant plasmid

9. AGCTTAGCGAT AGCTTAGCGAT TCGAATCGCTA TCGAATCGCTA AATCGC Tools for Producing Recombinant DNA Probe: sequence of DNA that is complementary to the gene of interest; Used to locate a copy of the gene by hybridization Denature DNA by heating Add Probe Probe Binds to gene

10. Tools for Producing Recombinant DNA Library: collection of DNA sequences from one donor that can be screened by a probe to detect the gene of interest • Genomic library = all of the sequences from the genome of a single organism • cDNA library= complementary DNA, made using mRNA as a template, used to study transcribed sequences

11. Building aGenomic Library

12. mRNA Reverse transcriptase TTTTTTTT AAAAAAAA DNA polymerase TTTTTTTT AAAAAAAA nuclease nuclease Exon 1 Exon 2 Exon 3 G AAAAAAAA…… Producing cDNA Molecules TTTTTTTT One strand of cDNA Second strand of cDNA To produce a cDNA library, each cDNA from the same cell type would be inserted into a vector cDNA

13. Using a Probe to Screen a Library for a Gene of Interest

14. Applying Your Knowledge • Probe • Clone • Plasmid • Library • Restriction Enzyme • Which one best describes • An enzyme that cleaves DNA at specific sequences? • A sequence of DNA that is complementary to the gene of interest? • A collection of DNA molecules used to find a gene of interest? • A small, independently replicating DNA molecule?

15. Biotechnological Methods: PCRhttp://www.dnalc.org/ddnalc/resources/pcr.html • PCR = Polymerase Chain Reaction • Amplifies a specific region in the DNA • Used for identification, especially if the amount of DNA is small • Uses repeated cycles of heating to denature DNA and cooling to synthesize new DNA • Involves the use of ---Taq polymerase (withstands heat) ---primers to begin synthesis

16. Polymerase Chain Reaction:One PCR Cycle 90 °C 50 °C 72 °C Taq Polymerase Primer OriginalDouble-helixDNA SeparateDNAStrands Primers &Taqpolymerasebind DNAsynthesized

17. Polymerase Chain Reaction:Multiple PCR Cycles 2 copies 4 copies 8 copies DNAfragmentto beamplified

18. DNA Fingerprinting with PCR

19. Biotechnological Methods: RFLP • RFLP Analysis • Restriction Fragment Length Polymorphism • Use of a probe to identify specific DNA fragments derived from restriction enzyme digestion • Shows variations in sizes of fragments between different individuals

20. Separation of Restriction Fragments by Size

21. DNA separated by size is transferred from agarose gel to filter DNA on filter is exposed to probe to detect complementary sequences. Southern Blotting for RFLP Analysis

22. RFLP Analysis used for Paternity Testing X X X X X X X X

23. RFLP Analysis in Forensics CSRCIE MNEE 1 2 3 4 5 6 7 Suspects Suspects

24. RFLP Analysis in Genetic Testing On the basis of this analysis, the genotype of the fetus is 1. AS 2. AA 3. SS 4. Unknown