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Molecular Biology (1)

Molecular Biology (1). Mamoun Ahram, PhD Second semester, 2015-2016. Pay attention to this…. You are allowed a total absence of 15% of total credit hours (7 days) without excuses and 20% (9 days) with excuses.

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Molecular Biology (1)

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  1. Molecular Biology (1) Mamoun Ahram, PhD Second semester, 2015-2016

  2. Pay attention to this… • You are allowed a total absence of 15% of total credit hours (7 days) without excuses and 20% (9 days) with excuses. • If you exceed this limit, you will fail the class regardless of the 10% score. The two are independent of each other. • No points will be subtracted from your 10% for the first 3 absences.

  3. Content for molecular biology

  4. Resources • This lecture • Campbell and Farrell’s Biochemistry, Chapter 9, 13 (pp. 381-382)

  5. Nucleic acids • The primary structure of nucleic acids is the order of bases in the polynucleotide sequence. • The secondary structure is the three-dimensional conformation of the backbone. • The tertiary structure is specifically the supercoiling of the molecule.

  6. Chemical composition and bonds Glycosidic bond This is why they are acidic Ribose vs. deoxyribose • Positively charged ions (Na+ or Mg2+) and peptides with positively charged side chains can associate with DNA • Eukaryotic DNA, for example, is complexed with histones, which are positively charged proteins, in the cell nucleus.

  7. Nitrogenous bases Glycosidic bond

  8. Nucleotides vs. Nucleosides

  9. Nucleotides vs. Nucleosides

  10. Nucleic acid polymer • A letter d can be added to indicate a deoxyribonucleotide residue. • for example, dG is substituted for G. • The deoxy analogue of a ribooligonucleotide would be d(GACAT).

  11. DNA structure • Specific base-pairing • A = T; G = C; Pur = pyr • Complementary • A double helix • Backbone vs. side chains • Antiparallel • Stable • Flexible • Groovings • Stability vs. flexibility

  12. Base pairing

  13. DNA forms • B-DNA • The principal form of DNA. • Right-handed • Base pairs are perpendicular. • A-DNA • 11 base pairs per turn • Base pairs lie at an angle • Right-handed. • Wider than B-DNA • Z-DNA • Left-handed • Occurs when alternating purine–pyrimidine and sequences with methylated C • Narrower than B-DNA

  14. DNA structure • Specific base-pairing • A = T; G = C; Pur = pyr • Complementary • A double helix • Backbone vs. side chains • Antiparallel • Stable • Flexible • Groovings • Stability vs. flexibility

  15. DNA coiling • The bacterial circular DNA can be coiled by rotating it clockwise (negative supercoiling) or counterclockwise (positive supercoiling). • Supercoils can be added or removed by enzymes called topoisomerases: • Class I topoisomerases cut the phosphodiester backbone of one strand of DNA. • Class II topoisomerases cut both strands of DNA. • In bacteria, it is called DNA gyrase.

  16. In eukaryotes… • In eukaryotes, DNA coiling is utilized for DNA packaging and regulation of gene activity. • Eukaryotic DNA is complexed with a number of proteins. • Chromatin = DNA molecule + proteins • The principal proteins in chromatin are the histones.

  17. Nucleosomes • The protein core is an octamer (two molecules of histones H2A, H2B, H3, and H4). • A linker DNA/spacer region connects the octamer-DNA complexes. • A nucleosome consists of DNA wrapped around a histone core. • H1 is bound to the the octamer and wrapped DNA (a chromatosome). • Histones are positively cahrged facilitating DNA interaction and charge neutralization.

  18. Light absorbance of nucleic acids • Aromatic pyrimidines and purines can absorb UV light • The peak absorbance is at 260 nm wavelength • The absorbance of 260 nm (A260) is constant • dsDNA: A260 of 1.0 = 50 ug/ml • ssDNA: A260 of 1.0 = 30 ug/ml • ssRNA: A260 of 1.0 =40 ug/ml Reason for ss vs. ds absorbance: • Stacked bases, vs. unstacked bases hyperchromicity What is the concentration of a double stranded DNA sample diluted at 1:10 and the A260 is 0.1? DNA concentration = 0.1 x 10 x 50 µg/ml = 50 µg /ml

  19. Observation of denaturation • The transition temperature, or melting temperature (Tm). • Factors influencing Tm • G·C pairs • Hydrogen bonds • Base stacking • pH • Salt and ion concentration • Destabilizing agents (alkaline solutions, formamide, urea)

  20. Central dogma of biology • Genetic information must be preserved via DNA replication. • Information must be translated into action makers (proteins) via transcription and translation. • RNA Sequence is dictated by DNA sequence.

  21. RNA • Consist of long, unbranched chains of nucleotides joined by phosphodiester bonds between the 3’-OH of one pentose and the 5’-OH of the next • The pentose unit is β-D-ribose (it is 2-deoxy-D-ribose in DNA) • The pyrimidine bases are uracil and cytosine (they are thymine and cytosine in DNA) • In general, RNA is single stranded (DNA is double stranded).

  22. Types of RNA

  23. Hybridization • DNA from different sources can form double helix as long as their sequences are compatible (hybrid DNA)

  24. Techniques • Gel electrophoresis • Hybridization techniques

  25. Gel electrophoresis • The length and purity ofDNA molecules can be accurately determined by the gel electrophoresis wells - - Direction DNA travels +

  26. Resources • http://www.personal.psu.edu/pzb4/electrophoresis.swf • http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html • http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html

  27. Detection Size Standard • The DNA molecules of different lengths will run as "bands“ • DNA is stained (that is, colored) with a dye (ethidium bromide) or radioactively labeled (32P) Sample 1 Sample 2 - 1000 bp 850 bp 750 bp 600 bp 200 bp 100 bp +

  28. Hybridization techniques • Hybridization reactions can occur between any two single-stranded nucleic acid chains provided that they have complementary nucleotide sequences • Hybridization reactions are used to detect and characterize specific nucleotide sequences

  29. Probes • A probes is a short sequence of single stranded DNA (an oligonucleotide) that is complementary to a small part of a larger DNA sequence • Hybridization reactions use labeled DNA probes to detect larger DNA fragments

  30. Southern blotting • This technique is a combination of DNA gel electrophoresis and hybridization • Used to detect: • the presence of a DNA segment complementary to the probe • the size of the DNA fragment

  31. 1 2 1 2 1 2 : probe Southern blot Electrophoresis

  32. Restriction endonucleases, RFLP, and gene cloning

  33. Resources • This lecture • Campbell and Farrell's Biochemistry, Chapter 13, pp. 357-367; 382-384

  34. Endonucleases • Enzymes that degrade DNA within the molecule rather than from either end (exonucleases) • Restriction endonucleases: Enzymes that recognize and cut (break) the phosphodiester bond between nucleotides at specific sequences (4- to 8-bp restriction sites) generating restriction fragments. • Type II restriction endonucleases: Always cleave always at the same place generating the same set of fragments • EcoRI (isolated from E. coli) cuts at 5'-GAATTC-3‘ • Some enzymes cut DNA at related sites • HinfI (from Haemophilus influenzae) recognizes 5'-ANTC-3' ('N' is any nucleotide) • Cuts at 5'-AATC-3', 5‘-ATTC-3', 5‘-AGTC-3' and 5'-ACTC-3’

  35. Biological purpose of restriction endonucleases • They are present in bacteria to protect them from bacteriophages that infect bacteria by transferring their DNA into them restricting their growth.

  36. Types of cleavages • Restriction enzymes cut DNA in two different ways: • Blunt: enzymes cut at the same position on both strands giving a blunt ended fragments • Staggered (off-center): enzymes cut the two DNA strands at different positions generating sticky or cohesive ends • The DNA fragments have short single-stranded overhangs at each end.

  37. 5’ vs. 3’ overhangs

  38. Palindromic sequence • The sequences recognized by restriction endonucleases—their sites of action—read the same from left to right as they do from right to left (on the complementary strand).

  39. DNA ligase • Covalently joins DNA ends (example, restriction fragments) • Catalyzing the formation of 3’→ 5’ phosphodiester bonds between the 3-hydroxyl end of one strand and the 5-phosphate end of another strand

  40. Advantage of restriction endonucleases • Restriction fragment length polymorphism (RFLP) • Cloning

  41. DNA polymorphisms • Individual variations in DNA sequence may create or remove restriction-enzyme recognition sites generating different restriction fragments • Remember: our cells are diploid (alleles can be homozygous or heterozygous) • What is an allele?

  42. Restriction fragment length polymorphism • The presence of different DNA forms in individuals generates a restriction fragment length polymorphism, or RFLP. • These can be detected by • Gel electrophoresis • Southern blotting

  43. Example

  44. RFLP in the clinic • RFLP can be used as diagnostic tools. • For example, if a mutation that results in the development of a disease also causes the generation of distinctive RFLP fragments, then we can tell • if the person is diseased as a result of this mutation • from which parent this allele is inherited

  45. Disease detection by RFLP

  46. Think!! What would you see in a gel?

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