Mamoun Ahram, PhD Second semester, 2015-2016 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. 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.

Content for molecular biology Nucleic acid structure Restriction endonucleases, RFLP, DNA fingerprinting, recombinant technology, and DNA cloning DNA replication DNA sequencing and PCR DNA mutations DNA repair RNA transcription and its regulation Protein synthesis and its regulation Cancer: a molecular perspective

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

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.

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.

Nitrogenous bases Glycosidic bond

Nucleotides vs. Nucleosides

Nucleotides vs. Nucleosides

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).

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

Base pairing

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

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

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.

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.

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.

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

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)

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.

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).

Types of RNA

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

Techniques Gel electrophoresis Hybridization techniques

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

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

Detection 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) Size Standard Sample 1 Sample 2 - 1000 bp 850 bp 750 bp 600 bp 200 bp 100 bp +

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

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

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

1 2 1 2 1 2 : probe Electrophoresis Southern blot

Restriction endonucleases, RFLP, and gene cloning

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

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’

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.

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.

5’ vs. 3’ overhangs

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).

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

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

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?

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

Example

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

Disease detection by RFLP

Think!! What would you see in a gel?

Example 1: Disease detection by RFLP (sickle cell anemia)

Normal/ carrier Normal Diseases Father Mother Son1 Son2 Son3

Example 2: Disease detection by ASO (Cystic fibrosis) ASO: Allele-specific oligonucleotide

Example 3: Paternity testing

Example 4: Forensics

Cloning Cloning means that you make several copies of one thing. A clone is a genetically identical population, whether of organisms, cells, viruses, or DNA molecules. Every member of the population is derived from a single cell, virus, or DNA molecule.

How do we clone a DNA molecule? a DNA fragment of interest is inserted into a DNA carrier (called a vector) that can be replicated. The resulting DNA molecule is what is known as a recombinant DNA molecule.

Using plasmids as vectors Bacterial plasmids are considered excellent vectors. These are bacterial circular DNA that is not part of the main circular DNA chromosome of the bacterium. A plasmid exists as a closed circle and replicates independently of the main bacterial genome.

Features of plasmids Can replicate Most plasmid vectors contain at least three essential parts required for DNA cloning: Can replicate Can be selected for/against by an internal drug-resistance gene (selectable marker) Can inset a foreign DNA fragment

Making of recombinant DNA Both DNA fragments (the DNA to be cloned and a vector) are cut by the same restriction endonuclease that makes sticky-ended DNA fragments When mixed, they will bind to each other

Cell clones Cloned DNA