MOLECULAR BIOLOGY Applied Sciences Division, Red River College

1. MOLECULAR BIOLOGY Applied Sciences Division, Red River College David Blicq http://atrey.karlin.mff.cuni.cz/~ofb/frames.html

2. Classic Reference Articles: • “The Polymerase Chain Reaction”, Nancy Smyth Templeton – Diagnostic Molecular Pathology 1(1): 58-72, 1992 • “Molecular Diagnostics of Infectious Diseases”, Yi-Wei Tang et al – Clinical Chemistry 43:11 2021-2038, 1997 • “Gene Cloning – An Introduction” T.A. Brown, Van Nostrand Publishers 1987

3. What is Molecular Biology? • Really an in-depth study of “life technology” • Involves complex chemical interactions of 1000’s of different types of molecules found in any cell! • How is it organized? DNA is the “Master Molecule”: Structure of DNA  information to create / direct the chemical machinery of life • What is “Molecular Genetics”? The study of the flow and regulation of the genetic information of DNA / RNA

4. A Hybrid Discipline: • Physics – energy changes and supporting math • Physical Chemistry – the many reactions • Microbiology – observation of single cells • Biochemistry – biochemical components and reactions • Genetics – the information behind life

5. History • Started with observation of structure of living organisms • Now examines the DNA / info controlling living systems

6. History • 1859 – Darwin – looks at changes in population • 1865 – Mendel – traits inherited by individual organisms • 1902 – Sutton – chromosome behaviour in cells • 1941 – Beadle and Tatum – biochemical interactions in cells • 1944 – Hershey and Chase – DNA interactions in cells • 1953 – Watson and Crick – molecular structure of DNA

7. 1956 - DNA is made artificially. • 1969 - The first single gene is isolated. • 1970 - The first artificial gene is made. • 1973 - Genetic engineering begins with the ability to insert genetic material. • 1976 - An artificial gene is inserted into a bacterium and, for the first time, works normally. • 1978 - Bacteria are engineered to produce insulin. • 1981 - A gene is transferred from one animal species to another. • 1984 - genetic fingerprinting developed. • 1984 - gene experiment to try to treat a 4-yr-old girl. • 1993 - Mice are cured of cystic fibrosis via gene therapy. • 2001 - The first draft of the human genome published.

8. History - Darwin • “Natural Selection” (over huge periods of time) selects for the “fittest” version of the life form • Arises as “best able to feed / mate” have offspring with strong characteristics • Occasionally get “random” physical changes that really help • Winning / fittest characteristics continue in offspring

9. History – Mendel(Austrian Monk) • Sought to explain how “winning” traits passed between generations • He related outward appearance (phenotype) to genetic makeup (genotype) • Determined Dominant and Recessive genes • Each parental cell has only one type • Combination of parental info is random! (Cc x Pp  CP cP Cp cp (four combos)

10. DNA Cloning: • Other names: “Recombinant DNA Technology” or “Genetic Engineering” • Idea – pick up a “desirable” characteristic and copy / clone it • Extreme version? – clone whole organism (ie. Dolly the sheep) • Typically focus on one characteristic / set of genes at a time

11. Recombinant DNA products being used in human therapy • insulin for diabetics • factor VIII for males suffering from hemophilia A • factor IX for hemophilia B • human growth hormone (GH) • erythropoietin (EPO) for treating anemia • three types of interferons • several interleukins • granulocyte-macrophage colony-stimulating factor (GM-CSFstimulating bone marrow • tissue plasminogen activator (TPA) dissolving blood clots • adenosine deaminase (ADA) for treating immunodeficiency • angiostatin and endostatin for trials as anti-cancer drugs • parathyroid hormone

12. So you want to tinker with life itself?5 General steps to clone a DNA sequence: • Cut DNA at precise locations (restriction endonucleases chop specific sites) • Join 2 fragments together (enzyme DNA ligase joins DNA pieces) to get “recombinant DNA” (composite molecules of DNA containing covalently-linked segments. • Incorporate DNA into “cloning vectors” such as plasmids (small DNA rings) or viral DNA • Transfer DNA (from test tube / cloning vector) into host cell to copy / reproduce the DNA or amplify via PCR • Have a method to identify / isolate “host cells” containing newly-made “recombinant DNA”

13. : http://www.troy.k12.ny.us/thsbiology/skinny/skinny_genetics.html

14. Basic Steps in Gene Cloning + + Host cell Transformed host cell Fragmented genomic DNA or cDNAs from chosen resource Recombinant DNA molecules Vector Amplification of recombinant molecule Host cell division 1 ‘gene’ purified in a clone Numerous cell divisions  CLONE Colonies of transformed host cell clones growing on solid medium But how many individual clones needed to represent the entire genomic DNA or expressed genes of the resource?

15. Deftns: Restriction / Modification of DNA • Bacteriophage: a virus capable of replicating within a bacterial cell • Restriction: the chopping / fragmenting of DNA • Modification: change / modify infecting DNA to make it resistant to restriction. Usually accomplished by methylating (+ CH3) or glycosylating (+ sugar) specific bases at specific sites

16. Restriction: Chopping DNA http://alpha.furman.edu/~lthompso/bgy30/dnatech/ttVectors.html

17. Example Restriction / Modification: • Bacteriophage T4 has no cytosine (has HMC / hydroxymethylcytosine) • Early in viral infection, the T4 DNA codes for two endonucleases which chop the host DNA into fragments (at cytosine site) • Fragments containing cytosine are then modified by T4 enzymes to stop further restriction. • Generally – virus enters cell, takes over genetic machinery and makes copies of itself (at the expense of the host)

18. Example: Restriction / Modification • ECO R1 (a restriction endonuclease from E.coli) cleaves DNA with specific sequence: 5’…GAATTC…3’ • Modification enzyme ECO R1 Methylase methylates one Adenine (A) on each strand within the ECO R1 (palindromic) sequence: 5’…GAATTC…3’ 3’…CTTAAG…5’ 3. This blocks further restriction / chopping

19. Specificity of Restriction Endonucleases: • R.E. are found in a wide range of bacterial species • Biological function? To recognize and cleave / destroy foreign DNA such as infecting viruses • A cell’s own DNA is protected by having the recognition sequences methylated • 3 General Types of R.E. Class I, Class II, and Class III

20. Classes of Restriction Endonucleases: Class I and III • Tend to be large, multi-subunit complexes • Have both endonuclease and methylase activities • Type I – cleave DNA at random sites up to 1000 bp (base pairs) from recognition sequence • Type III – cleave DNA at ~25bp from recognition site • Both types – require energy (in form of ATP)

21. Class II Restriction Endonucleases • Tend to be much simpler • Cleave DNA at / within recognition sequence • Recognize short, specific palindromes • Forms either “Blunt” (strands cut across from each other) or “Cohesive” ends (cuts DNA at a point several bp away from complementary bases http://www.blc.arizona.edu/marty/411/Modules/transition.html

22. Class II RE –Cohesive (“Sticky”) Ends=- --= http://www.uic.edu/classes/phar/phar331/lecture5/

23. Class II RE – Blunt Ends (= =) http://www.uic.edu/classes/phar/phar331/lecture5/

24. Enzymes used in Recombinant DNA Technology: • Type II RE – cleave DNA at specific sequences • DNA Ligase – joins two fragments • DNA Polymerase – fills gaps by adding nucleotides • Reverse Transcriptase – make DNA copy of RNA • Polynucleotide Kinase – add phosphate to 5’ end • Terminal Transferase – add “tails” to 3’-OH end • Exonuclease III - remove nucleotides fr5om 3’ end • Bacteriophage lambda – remove nucleotides from 5’ end to expose singled-stranded 3’ends • Alkaline Phosphatase – removes terminal Pi from 5’3’ ends

25. Specific Class II R.E. • ECO RI (E.coli RY13) - cohesive ends • BAM H1 (B.amyloliquifaciens) - cohesive • HIND III (H.influenzae Rd) - cohesive • Sma I (Serratia marcesens) - blunt ends • HIND II (H. influenzae Rd) - blunt ends • Hae II (H.aegyptius) - cohesive

26. Specific Cloning Vectors • Plasmids • Bacteriophage • Cosmids • Yeast artificial chromosomes (YACs) • Bacterial artificial chromosomes (BACs) Deftn: “Amplify”- to increase the number of (inserted DNA segments).

27. Plasmids • Small, circular DNA molecules that replicate separately from the host chromosome (Xm) • In nature, usually 5,000 – 400,000 bp in length • Plasmids can be introduced into cells via “Transformation”

28. Transformation • Get “new” DNA into host cells • Incubate cells + DNA in CaCl2 at 0ºC • Apply heat shock (raise temp to 37-43ºC) • A few cells in the host bacteria population take up the plasmid DNA • Must have a method of isolating the few transformed cells • An inexpensive way of amplifying DNA

29. Characteristics of a “Good Plasmid” (i.e. pbr322) • Makes at least 10-20 copies per cell • 2 genes that provide resistance to antibiotics present • Has several recognition sequences for different R.E. so it can be easily cut and foreign DNA inserted • Small size – easier to enter cells (bigger plasmids have reduced transformation efficiency)

30. Example Plasmid – PBR322 http://www.brunel.ac.uk/depts/bl/project/genome/moltec/vectors/pbr322.htm

31. http://www3.kumc.edu/jcalvet/bioc801i/sld010.htm

32. http://athena.uwindsor.ca/units/biochem/web/protei .nsf/18e87328064218268525698 30050331b/7a371e9af805f74e85256a4f00538021/$FILE/11

33. http://athena.uwindsor.ca/units/biochem/web/protein.nsf/18e8732806421826852569830050331b/7a371e9af805f74e85256a4f00538021/$FILE/11http://athena.uwindsor.ca/units/biochem/web/protein.nsf/18e8732806421826852569830050331b/7a371e9af805f74e85256a4f00538021/$FILE/11

34. Bacteriophage λ (Phage) Vector • A bacteriophage is a virus capable of replicating in a bacterial (host) cell – example: “Lambda” • Can clone larger DNA fragments than plasmid vectors • 1/3 of phage genome (DNA) is non-essential (therefore can be replaced with foreign DNA) • Size specific – only DNA ~ 40-50 kbp will be inserted into phage vector

35. http://www.urmc.rochester.edu/smd/mbi/grad2/phage01.html

36. Cosmid Vectors • Overcome insert size limit of plasmid and bacteriophage vectors (15-20 KB) • Cosmid vectors are plasmid / bacteriophage hybrids • Contain cohesive site (cos) of phage (required for in vitro packaging) • Accept inserts of 35 to 45 kb

39. Different Cloning Vectors for Different Applications Cloning Vector Standard high copy number plasmid Bacteriophage  insertion Bacteriophage  replacement Cosmid Bacteriophage P1 PAC (P1 Artificial Chromosome) BAC (Bacterial Artificial Chromosome) YAC (Yeast Artificial Chromosome) Insert Size (kb) ≤ 10 ≤ 10 9-23 30-44 70-100 130-150 ≤ 300 0.2-2000

41. Structure of RNA • RNA is always synthesized as a single strand • RNA twists into a R.H. helix • Unlike DNA, there is no regular structure! • Can have single strands, bulges, hairpins, internal loops, hairpins, etc. • Examination of 3-dimensional RNA structure is only just beginning  lots of good research ahead

42. RNA Structure http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/Nucleic_Acids/nucacid_structure.html

43. Basic Chemistry of Nucleic Acids • DNA is a good “warehouse” for genetic information because it is very stable! • Chemical changes that do occur are very slow (when there is no catalyst) this is good don’t want random changes • Even slow/small changes in DNA can have a profound physiological impact!

44. Basic Chemistry of Nucleic Acids • Aging and carcinogenesis (cancer development) may be linked to slow, irreversible changes in DNA structure • Appropriate structure / chemical changes include strand separation (for DNA replication or transcription)

45. Denaturation of Double Helix DNA and RNA • At pH 7.0 (physiological pH) DNA is very vicous at room temperature. • At extremes of pH / temperature viscosity drops (so DNA is undergoing a physical change – similar to pH/temp effects on proteins) • Denaturation (melting) of DNA involves: • disruption of hydrogen bonds between b.p. • disruption of hydrophobic interactions between stacked bases • What happens? Double helix unwinds to form two single strands

46. Denaturation of DNA http://www.cas.muohio.edu/~wilsonkg/genetics/Scimeth/SciMeth_W_C/DNA_Properties/melt/melt.htm

47. Annealing / Renaturation: • Requires at least a dozen (or more) residues intact (a fast one-step process) • Completely separate strands? Re-forms much slower (ie. Complementary b.p. must find each other through random collisions) • Once ~12 bases are paired the annealing is quick – kind of like a molecular “zipper”

48. Denaturation at Characteristic Temperatures • Bacterial and viral DNA (in solution) denature at specific temperatures (when slowly heated). • The shift from double-stranded helix to denatured single strands can be monitored by absorbance: when DNA separate, u.v. absorbance increases!

49. Strand Separation / Melting: http://www.biologie.uni-hamburg.de/b-online/e21/14.htm

50. Diff. Species = Diff. G/C Content: http://www.massey.ac.nz/~wwbioch/DNA/DS-DNA/DNAtext.htm