Biotechnology Research

1. Biotechnology Research BIT 220 MCCC Chapter 10

2. Terminology Molecular Biology/Genetics: the study of gene structure and function at the molecular level Molecular Biotechnology: the ability to transfer specific units of genetic information from one organism to another Recombinant DNA Technology = gene splicing = genetic engineering =gene transplantation = gene cloning =molecular cloning

3. Genetics • Science of Heredity • Explains similarities and differences between organisms • Two Branches • Classical (Mendelian) • cells contain pairs of ‘factors’ which determine physical characteristics • these factors segregate during meiosis, independently • Molecular

4. DNA/RNA Molecular Basis of Life Central Dogma DNA RNA protein control Transcription Translation DNA - Deoxyribonucleic Acid RNA - Ribonucleic Acid DNA DNA Replication (mitosis,meiosis)

5. DNA Structure Replication Gene Expression Mutation

6. PROPERTIES OF DNA 1.Replicate Genotypic function 2.Mutate – chemically change and transmit these changes to future generations - if mutant in germ line evolutionary function 3.Gene expression Direct the synthesis of proteins phenotypic function

7. 1. Miescher DNA • 1868 isolated DNA from pus cells (NUCLEIN) • 2. Frederic Griffith 10.1 and 10.2 • 1928 ‘transforming principle’ in pneumococcus • 3. Oswald Avery, Colin MacLeod, Maclyn McCarty 10.3 • 1944 DNA was the active substance in the heat-killed S strain extracts because DNases destroyed the activity while RNases and proteases did no.

8. 4. Chargaff the molar concentration of A = T and G = C which led to the discovery of base pair complementarity. 5. Wilkins and Franklin 10.10 X-ray crystallographs (diffraction patterns) of DNA -Review DNA structure: Figures 10.6, 10.7, 10.8, 10.11, 10.12, 10.13, Table 10.3 6. Watson and Crick deduced a two-stranded structure wrapped in a right-handed helix with the bases internal, the phosphates external, and an internal repeating subunit separated by 0.34 nm

9. Smallest unit of nucleic acid • Three components • 1. sugar • 2. phosphate • 3. nitrogenous base Nucleotide

10. Other Nomenclature Nucleoside : SUGAR and BASE (NO PHOSPHATE) BASES NUCLEOSIDES NUCLEOTIDES Adenine Deoxyadenosine Deoxyadenosine 5’-triphosphate Guanine Deoxyguanosine “” (dATP) Cytosine Deoxycytidine “” Thymine Deoxythymidine “”

11. Double Helix 2 strands wrapped around one another SPIRAL STAIRCASE Allows replication Allows permanence Bases on one side of helix are complementary to other strand What draws two strands together? 1. Bases hydrogen bond 2. Bases are hydrophobic

12. DNA Structure A. Sugar-Phosphate Backbone covalent bonds between S and P phosphodiester linkages B. Stairs Sugar covalently bound to base Bases hydrogen bound to each other A::T C:::G

13. DNA Antiparallel Strands One strand runs 3’C (OH of sugar) to 5’ C (phosphate); The other strand 5’ to 3’

14. Alternate forms A 11 bp per turn/ right-handed B 10.4 base pairs per turn/ right handed FOUND IN VIVO - physiological DNA form Z 12 bp per turn/left handed

15. Supercoiling In vivo negatively supercoiled (underwound) Introduced when one or both strands nicked and strands rotate around one another Figures 10.14 and 10.15

16. Chromosome Structure Composed of DNA (RNA viruses) and proteins single piece of nucleic acid Prokaryotic (bacteria and virus) single chromosome/ 3000 genes E coli condenses DNA into loops 10.16 no introns all single copy DNA monoploid Eukaryotic 1000 times more DNA than bacteria noncoding regions introns 30-85% single copy most diploid

17. Eukaryotic Chromosomes Chromatin a. DNA b. non-histone proteins c. histone proteins: 10.23 and10.24 DNA wraps around dimers of each H2a, H2b, H3, and H4 forming nucleosome H1 sits outside nucleosome

18. DNA Compaction 10.27 Level 1 Nucleosome Level 2 supercoiling of nucleosomes Level 3 Scaffold composed of non-histone proteins packing during metaphase (most condensation)

19. Centromeres Figures 10.29 and 10.30 interchangeable among chromosomes 110-120 bp in length. Telomeres Figure 10.31 contain special repeated DNA sequences that enable the ends of the chromosomes to be replicated, inhibit their degradation by DNA degrading enzymes, and prevent fusion with other chromosomes. In vertebrates, the TTAGGG repeat is highly conserved and in humans 500-3000 repeats occur in telomeres which gradually shorten with age.

20. Eukaryotic DNA contains excess DNA, up to 50% or more, that does not code for proteins and comprises families of highly repeated sequence elements or repetitive DNA (satellite DNA) • three classes of DNA • 1. unique or single-copy • 1-10 copies per haploid genome • genes that encode proteins • regulatory sequences • 2. moderately repetitive • 10-100,000 copies • transposable elements (jumping genes) • rRNA • histone • ribosomal genes • 3. highly repetitive • >100,000 copies • telomeres • centromeres • unknown function

21. Types of repeats LINEs - Long Interspersed Nuclear Elements SINEs - Short Interspersed Nuclear Elements LTRs - Long terminal repeats DNA transposons (transposable elements)

22. Denaturation/Renaturation D: Two strands of DNA separate (Melt) R: Two strand hydrogen bond through complementary bases (Annealing) The kinetics of these processes will indicate the amount of repetitive DNA Higher the copy number; the faster the renaturation occurs

23. Hybridization Radioactive-labelled sequences Nucleic Acid probe contains radioactive isotope this isotope emits charged particles as it decays these charged particles captured on emulsion (film) exposed silver halides produce back spots Fluorecently-labelled sequences Tech Sidelight