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Chapter 20

Chapter 20. Techniques of Molecular Biology. The methods of molecular biology depend upon and were developed from an understanding of the properties of biological macromolecules themselves. Part I NUCLEIC ACID. NUCLEIC ACIDS. DNA and RNA separation by gel electrophoresis.

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Chapter 20

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  1. Chapter 20 Techniques of Molecular Biology

  2. The methods of molecular biology depend upon and were developed from an understanding of the properties of biological macromolecules themselves.

  3. Part I NUCLEIC ACID

  4. NUCLEIC ACIDS DNA and RNA separation by gel electrophoresis • Principle: Linear DNA molecules migrate through the gel toward the positive pole with different rates when subject to an electrical field. • The DNA molecules can be visualized by staining the gel with fluorescent dyes, such as ethidium.

  5. NUCLEIC ACIDS • Two matrices: polyacrylamide and agarose. Plyacrylamide has more resoving power. • Pulsed-field gel electrophoresis for long DNAs (up to several Mb in length).

  6. According to RNA it is similar, however RNA sample should be treated with reagents ,e.g. glyoxal to prevent the formation of base pairs. NUCLEIC ACIDS

  7. Restriction Endonuleases Cleaves DNA Molecules at Particular Sites NUCLEIC ACIDS • Restriction enzymes recognize short target sequences and cut at a defined position within those sequences. • They can generate different ends: flush ends and staggered ends. • We use them to break large DNA into manageable fragments.

  8. NUCLEIC ACIDS Recognition sequences and cut sites of various endonucleases

  9. How we name them?? Take EcoRI for example: Eco: E. coli I: the first one

  10. Hybridization probes can identify electrophoretically separated DNA and RNA NUCLEIC ACIDS • Southern blot named after Edward Southern: • DNA fragments, generated by digestion of a DNA molecule by a restriction enzyme, are run out on an agarose gel. • Once stained, a pattern of fragments is seen. • When transferred to a filter and probed with a DNA fragment homologous to just one sequence in the digested molecule, a single band is seen, corresponding to the position on the gel of the fragment containing that sequence.

  11. NUCLEIC ACIDS One example of southern blot

  12. DNA Cloning NUCLEIC ACIDS • Some terms:DNA cloning; vector; insert DNA;library: a population of identical vectors that each contains a different DNA insert.

  13. NUCLEIC ACIDS • Characteristics of vector DNAs:1.an origin of replication2.a selectable marker3.sigle sites for one or more restriction enzymes.

  14. How to clone DNA in plasmid vectors: NUCLEIC ACIDS • A fragment of DNA , generated by cleavage with a certain restriction enzyme, is inserted into the plasmid vector linearized by the same enzyme. • The recombinant plasmid is introduced int o bacteria by transformation. • Cells containing the plasmid can be selected by growth on the antibiotic to which the plasmid confers resistance.

  15. Construction of a genomic DNA library: NUCLEIC ACIDS • Genomic DNA and vector DNA, digested with the same restriction enzyme, are incubated together with ligase • The resulting pool or library of hybrid vectors is then introduced into E. coli, and the cells are plated onto a filter placed over agar medium. • The filter is removed from the plate and prepared for hybridization.

  16. NUCLEIC ACIDS

  17. Construction of a cDNA library NUCLEIC ACIDS • Isolate mRNA • use reverse transcriptase to synthesize complementary DNA strand from mRNA, then use DNA Pol I to synthesize double stranded DNA. Clone these cDNAs into appropriate vector (usually plasmid or phage) • Use Oligo dT primer to hybridize to polyA tail of mRNA. Primer used by reverse transcriptase for extension. • Reverse transcriptase is a DNA polymerase which uses RNA as a template to synthesize complementary DNA. Cloned from RNA viruses.

  18. We should note that: NUCLEIC ACIDS • No introns cloned, nor regulatory sequences • Genes cloned in this method are only those that were expressed in the particular tissue mRNA was isolated from.

  19. NUCLEIC ACIDS

  20. NUCLEIC ACIDS After having constructed a DNA library, whether genomic or cDNA, we can use probes to find specific clones we are interested in.

  21. Site-directed mutagenesis NUCLEIC ACIDS Using site-directed mutagenesis the information in the genetic material can be changed. A synthetic DNA fragment is used as a tool for changing one particular code word in the DNA molecule. This reprogrammed DNA molecule can direct the synthesis of a protein with an exchanged amino acid.

  22. Polymerase Chain Reaction NUCLEIC ACIDS The Royal Swedish Academy of Sciences awards 1993’s Nobel Prize in Chemistry to: For more, clickhttp://nobelprize.org

  23. NUCLEIC ACIDS • for contributions to the developments of methods within DNA-based chemistry • for his invention of the polymerase chain reaction (PCR) method • for his fundamental contributions to the establishment of oligonucleotide-based, site-directed mutagenesis and its development for protein studies

  24. Let’s look into it in more details: NUCLEIC ACIDS • Denaturation at 94℃ : the double strand melts open to single stranded DNA, all enzymatic reactions stop . • Annealing at 54℃ :The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. • Extension at 72℃ :This is the ideal working temperature for the polymerase. The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template)

  25. NUCLEIC ACIDS

  26. How to determine the sequence of bases in a DNA molecule NUCLEIC ACIDS • The most commonly used method of sequencing DNA - the dideoxy or chain termination method - was developed by Fred Sanger in 1977 (for which he won his second Nobel Prize). The key to the method is the use of modified bases called dideoxy bases; when a piece of DNA is being replicated and a dideoxy base is incorporated into the new chain, it stops the replication reaction.

  27. The Nobel Prize in Chemistry 1980 NUCLEIC ACIDS For more, clickhttp://nobelprize.org

  28. Elements: NUCLEIC ACIDS • The DNA to be sequenced: in single-stranded form; as a template. • The four nucleotides • The enzyme DNA polymerase and a primer • A nucleotide analogue that cannot be extended and thus acts as a chain terminator

  29. NUCLEIC ACIDS Dideoxynucleotides used in DNA sequencing

  30. NUCLEIC ACIDS Train termination in the presence of dideoxynucleotides

  31. Mechanism: NUCLEIC ACIDS

  32. NUCLEIC ACIDS

  33. One example of fluorecent chain-terminating nucleotides: NUCLEIC ACIDS

  34. Sequencing Whole Genomes NUCLEIC ACIDS

  35. NUCLEIC ACIDS • First, the source clone is fragmented, producing a random mixture, and a random sub-clone is selected for sequencing by the Sanger method. • To ensure that that the whole source clone has been sequenced, this stretch of DNA must be sequenced numerous times to produce an ordered overlapping sequence. • Gaps in this process will occur where a sub-clone is not fully sequenced.

  36. Contigs: NUCLEIC ACIDS • Assemble the short sequences from random shotgun DNAs into larger contiguous sequences.

  37. NUCLEIC ACIDS Contigs are linked by sequencing the ends of large DNA fragments

  38. Genome-wide analyses NUCLEIC ACIDS • Animal genomes contain complex exon-intron structure, so it is more difficult to find protein coding genes.

  39. NUCLEIC ACIDS • A variety of bioinformatics tools are required to identify genes and determine the genetic composition of complex genomes. • A notable limitation of current gene finder programs is the failure to identify promoters • EST (expressed sequence tag) is simply a short sequence read from a larger cDNA.

  40. NUCLEIC ACIDS Gene finder methods: Analysis of protein–coding regions in Ciona

  41. Comparative Genome Analysis NUCLEIC ACIDS • Permits a direct assessment of changes in gene structure and sequence arisen during evolution. • Refines the identification of protein-coding genes within a given genome.

  42. What we have learned from comparative genome analysis NUCLEIC ACIDS • Synteny: conservation in genetic linkage, between distantly related animals.

  43. Part II PROTEINS

  44. Purification of proteins PROTEINS • To purify proteins we make use of their inherent similarities and differences. • Protein similarity is used to purify them away from the other non-protein contaminants. • Differences are used to purify one protein from another. Proteins vary from each other in size, shape, charge, hydrophobicity, solubility, and biological activity.

  45. ImmunoAffinity Chromatography PROTEINS

  46. Affinity Chromatography PROTEINS • column matrix has a ligand that specifically binds a protein • specialty affinity columns for binding recombinant proteins with certain "tags"

  47. Affinity Chromatography PROTEINS

  48. Ion Exchange Chromatography PROTEINS • proteins have charges due to amino acid side groups • bind to charged column matrix depending on their charge at a particular pH • anionic--negatively charged: phosphocellulose, heparin sepharose, S-sepharose • cationic--positively charged: DEAE-sepharose, Q-sepharose • elute bound proteins from column based on charge and displacement by salt or pH

  49. Ion Exchange Chromatography PROTEINS

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