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DNA AND Biotechnology - detailed lecture notes
Biochemistry/Lab (CHEM 3650)
Nova Southeastern University
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DNA AND Biotechnology Lecture Notes (Can be used for MCAT review too)
DNA Structure
Two chemically distinct forms of nucleic acids within eukaryotic cells: Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA). DNA and RNA are polymers of one another Bulk of DNA is found in chromosomes in the nucleus
Nucleosides and Nucleotides
DNA is a polydeoxyribonucleotide which is composed of many monodeoxyribonucleotides linked together. Nucleosides: composed of five-carbon sugar (pentose) bonded to a nitrogenous base and are formed by covalently linking the base C-1’ of the sugar o Carbon atoms in sugar are labeled with a prime symbol to distinguish them from the nitrogenous base Nucleotides: formed when one or more phosphate groups are attached to C-5’ of a nucleoside. o Often named according to the number of phosphates present E. – adenosine di-/triphosphate o High energy compounds since there is repulsion between the closely associated negative phosphate groups. Nucleic acids are classified according to the pentose they contain o If pentose is ribose, then the nucleic acid is RNA o If pentose is deoxyribose (2’- OH groups replaced by –H), then it is DNA
Sugar-Phosphate Backbone
Backbone of DNA is composed of alternating sugar and phosphate groups This determines the directionality of the DNA and is always read from 5’ to 3’ Backbone is formed as the nucleotides are joined by 3’-5’ phosphodiester bonds o Phosphate group link the 3’ carbon of one sugar to the 5’ phosphate group of the next incoming sugar in the chain Since phosphates carry a negative charge, DNA and RNA strands have an overall negative charge. Each strand of DNA has a distinct 5’ and 3’ end, which creates polarity within the backbone o The 5’ end of DNA will have an –OH or phosphate group bonded to C-5’ of the sugar o The 3’ end of DNA will have a free –OH on the C-3’ of the sugar. Base sequence of nucleic acid strand will always be written and read in the 5’ to 3’ direction o If written backwards, the ends must be labeled as “3’” and “5’” (e. – 3’–GTA-5’) o Position of phosphates may be shown (e. – pApTpG) o “d” may be used as shorthand for deoxyribose (e. – dAdTdG) DNA is generally double-stranded (dsDNA) and RNA is generally single-stranded (ssRNA)
Purines and Pyrimidines
Two families of nitrogen-containing bases found in nucleotides: purines and pyrimidines Overall, there are five common bases, but there may be exceptions in tRNA or in some prokaryotes and viruses Purines: Contain two rings in their structure. o Adenine (A) and Guanine (G). o Both are found in DNA and RNA Pyrimidines: contain one ring in their structure o Cytosine (C), Thymine (T), & Uracil (U) o Cytosine is found in both DNA and RNA, thymine is only in DNA and uracil is only in RNA These are examples of biological aromatic heterocycles. Aromatic describes an unusually stable ring system that adheres to the following rules o Compound is cyclic o Compound is planar o Compound is conjugated (has alternating single and multiple bonds, or lone pairs, creating at least one unhybridized p-orbital for each atom in the ring) o Huckel’s Rule: compound has 4n+2 electrons. Stability from aromatic compounds results from delocalized pi-electrons which are able to travel throughout the entire compound using available molecular orbitals Heterocycles are ring structures that contain at least two different elements in the ring o Both purines and pyrimidines contain nitrogen in their aromatic rings o Thus nucleic acids have exceptional stability
Histones
DNA that makes up a chromosome is wound around a group of small basic proteins called histones, which forms a chromatin. Five histone proteins in eukaryotic cells Two copies each of the histone proteins H2A, H2B, H3, and H4 from a histone core o 200 base pairs of DNA can wrap around this protein complex o Nucleosome: histone protein complex with its DNA base pairs Look like beads on a string The last histone protein H1, seals off the DNA as it enters or leaves the nucleosome Histones are an example of nucleoproteins: proteins that associate with DNA o Most others are acid-soluble and stimulate processes
Heterochromatin and Euchromatin
Chromosomes have a diffuse configuration during interphase of the cell cycle Cell will undergo DNA replication during the S phase of interphase and the DNA is uncondensed during this process to make it more accessible o Small percentage of chromatin remains compacted during interphase and is called heterochromatin Heterochromatin: appears dark under light and does not partake in transcription o Consists of DNA with highly repetitive sequences Euchromatin: dispersed chromatin and appears light under light microscopy o Contains active DNA
Telomeres and Centromeres
DNA replication cannot extend all the way to the end of a chromosome. As such, the end of each chromosome is lost during replication. o Solution to this is to have a repeating unit at the end of DNA Telomere: simple repeating unit at the end DNA in order to prevent lost information o TTAGGG o Some of the sequence is lost in each round of replication and can be replaced by the enzyme telomerase More highly expressed in rapidly dividing cells o Set number of replications possible, shortening of telomere contributes to aging o High GC content in telomere creates exceptionally strong strand attractions at the end of chromosomes Prevents unravelling of chromosome Centromere: region of DNA at the center of chromosome o Sites of constriction because they form noticeable indentations o Composed of heterochromatin (repeat sequences with high GC content which promotes strong attraction) o Functions to keep sister chromatids connected until microtubules separate the chromatids during anaphase.
DNA Replication
Strand Separation and Origins of Replication
Replisome or Replication Complex: set of specialized proteins that assist the DNA polymerases Origins of Replication: Points where DNA begins to unwind. Beginning of replication process Replication Forks: generation of new DNA proceeds in both direction, which creates forks on both sides of the origin. Bacterial chromosome is a closed, double stranded circular DNA with a single origin of replication. Eukaryotic replication must copy many more bases and is a slower process o Each eukaryotic chromosome contains one linear molecule of double-stranded DNA that has multiple origins of replication. This allows the chromosomes to duplicate efficiently o As replication forks move forwards towards each other, sister chromatids are created. Chromatids remain connected at the centromere.
Helicase: enzyme responsible for unwinding the DNA, generating two single-stranded template strands ahead of polymerase. Once opened, the unpaired strands want to hydrogen bond with other molecules (there are free purines and pyrimidines) o Proteins are required to hold the strands apart. Single-stranded DNA-binding proteins bind to the unraveled strand which prevents the reassociation of the DNA strands and prevents the degradation of DNA by nucleases Supercoiling: wrapping of DNA on itself as its helical structure is pushed further toward the telomeres during replication. o Occurs as helicase unwraps DNA. o This strains the DNA helix and increases the chances of strand breakage. DNA topoisomerases: introduces negative supercoils which alleviates the torsional stress o Work ahead of helicase and “nick (cut)” one or both strands. This allows relaxation and the cut strands are then resealed. Parental strands will serve as templates for the generation of new daughter strands.
o DNA polymerase & are assisted by PCNA protein. This assembles into a trimer to form a sliding clamp, that helps to strengthen the interaction between these DNA polymerases and the template strand
Replicating the Ends of Chromosomes
DNA polymerase cannot complete the synthesis of the 5’ end of the strand o Thus each time the DNA synthesis is carried out, the chromosome becomes shorter Telomeres are used to lengthen the time that cells can replicate and synthesize DNA before necessary genes are damaged. o Repetitive sequence with high GC-content, and located at tips of the chromosome.
DNA Repair
DNA can be damaged from exposure to chemicals or radiation If not repaired, the damaged DNA will be passed on to daughter cell And damage causes an increased risk of cancer
Oncogenes and Tumor Suppressor Genes
Cancer can result from mutated genes. These cells can proliferate excessively since they are able to divide without stimulation from other cells o Able to migrate by local invasion or metastasis. Allows migration to distant tissue by the bloodstream or lymphatic system Oncogenes: mutated genes that cause cancer o Primarily encode cell cycle-related proteins Proto-oncogenes: before oncogene genes are mutated o Abnormal alleles encode proteins that are more active than normal proteins which promotes rapid cell cycle advancement Antioncogenes: tumor suppressor genes (like p53 or Rb) that encode proteins that inhibit the cell cycle or participate in DNA repair processes. o These function to stop tumor progression
o Mutation of these genes result in the loss of tumor suppression activity which then promotes cancer o Generally, both alleles need to be inactivated since even one copy of the normal protein is usually enough to inhibit tumor formation.
Proofreading and Mismatch Repair
DNA polymerase is usually 100% accurate, but it does occasionally make errors Proofreading During synthesis, two double-stranded DNA molecules will pass through a part of the DNA polymerase enzyme for proofreading If the wrong bases are paired, the hydrogen bonds between the bases are unstable. This instability can be detected as the DNA passes through the specified part of polymerase. o Incorrect base is removed and replaced with correct one. o Enzyme determines which is the template strand by analyzing level of methylation. The template strand has existed in the cell for a longer period of time so it will therefore be more methylated. DNA ligase does not have proofreading ability as it closes the gaps between Okazaki fragments. Thus, it much more likely to have a mutation in the lagging strand as compared to the leading strand. Mismatch Repair The G 2 phase of the cell cycle is able to perform mismatch repair Enzymes are encoded by genes MSH2 and MLH1, which detect and remove errors introduced in replication that were missed during the S phase of the cell In prokaryotes, the enzymes that do a similar function are MutS and MutL
Nucleotide and Base Excision Repair
Cell machinery is able to recognize two specific types of DNA damage in the G 1 and G 2 cell cycle phases and fixes them through nucleotide or base excision repair Nucleotide Excision Repair UV light induces the formation of dimers between adjacent thymine residues in DNA. o These dimers interfere with DNA replication and normal gene expression and distort the shape of the double helix. Thymine dimers are eliminated from DNA by a nucleotide excision repair (NER) o This is a cut and patch process. o Specific proteins scan the DNA molecule and recognize the lesion because of a bulge in the strand o An excision endonuclease then makes cuts in the phosphodiester backbone of the damaged strand on both sides of the thymine dimer and removes the defective oligonucleotide. o DNA polymerase then fills in the by synthesizing DNA in the 5’ to 3’ direction (uses the undamaged strand as a template) o The cut in the strand is sealed by DNA ligase
Base Excision Repair Cytosine deamination is the loss of an amino group from cytosine and results in its conversion to uracil. This is usually caused by thermal energy being absorbed by DNA
o Bacteria are then grown in colonies and a colony containing the recombinant vector is isolated. Can be done by ensuring that the recombinant vector includes a gene for antibiotic resistance. Antibiotics can then be administered and all other colonies will be killed o Resulting colony can then be grown in large quantities o Bacteria can then be made to express the gene of interest or can by lysed to reisolate the replicated recombinant vector Restriction Enzymes (restriction endonucleases): enzymes that recognize specific double stranded DNA sequences o Sequences are palindromic (Read the same backwards and forwards) o These enzymes are isolated from bacteria (natural source) o Restriction enzyme can cut through the backbones of the double helix once a specific sequence is identified o Some produce offset cuts which yields sticky ends on fragments Advantageous when the restriction fragment is joined with a vector DNA since they will fit together perfectly. DNA vectors contain at least one sequence that can be recognized by restriction enzymes.
DNA Libraries and cDNA
DNA Libraries: large collection of known DNA sequences which could potentially equate to the genome of an organism. o Can consist of either genomic DNA or cDNA Genomic Libraries: contain large fragments of DNA and include both coding (exon) and noncoding (intron) regions of the genome o Genomic libraries Complementary DNA (cDNA) Libraries: constructed by reverse-transcribing processed mRNA o Lacks noncoding regions and only includes genes that are expressed in the tissue from with the mRNA was isolated o Sometimes called expression libraries o Only cDNA libraries can be used to reliably sequence specific genes and identify disease-causing mutation, produce recombinant proteins or produce transgenic animals
Hybridization
The joining of complementary base pair sequences. This can be DNA-DNA recognition or DNA-RNA recognition Uses two single-stranded sequences Polymerase Chain Reaction (PCR) Automated process that can produce millions of copies of DNA sequence without amplifying the DNA in bacteria. Used to identify criminals, family relationships, and disease causing bacteria/viruses
Requires: primers that are complementary to the DNA that flank the region of interest, nucleotides (dATP, dTTP, dCTP, dGTP), and DNA polymerase o Primer has high GC content which increases stability Also needs heat to cause the DNA double helix to denature o DNA polymerase from humans does not work at high temperature, so DNA polymerase from a bacterium (Thermus aquaticus) is used. During PCR, DNA of interest is denatured, replicated and then cooled to allow reannealing of the daughter strands with the parent strands. DNA is doubled each cycle, and can be repeated as desired. Gel Electrophoresis and Southern Blotting Gel electrophoresis is used to separate macromolecules like DNA and proteins, by charge and size. All DNA strands will migrate towards the anode of an electrochemical cell since all DNA molecules are negatively charged Preferred gel is agarose gel. The longer the DNA strand, the slower it will migrate in the gel Electrophoresis is often used while performing a Southern Blot: used to detect the presence and quantity of various DNA strands in a sample o DNA is cut by restriction enzymes and then separated by gel electrophoresis o DNA fragments then transferred to a membrane (retain their separation) o Membrane is then probed by many copies of a single-stranded DNA sequence Probe will bind to its complementary sequence to form double-stranded DNA Probe is labeled to indicate the presence of a desired sequence
DNA Sequencing
Sequencing reaction requires template DNA, primers, DNA polymerase, and all four deoxyribonucleotide triphosphates o Dideoxyribonucleotide is a modified base that is also added at lower concentrations. Contain a hydrogen at C-3’ rather than on the hydroxyl groups ddATP, ddCTP, ddGTP, ddTTP o When the modified base is incorporated, the polymerase cannot add to the chain Eventually the sample will contain many fragments that each terminate with a modified base o As many fragments as the number of nucleotides in the desired sequence Fragments can then be separated by gel electrophoresis and the last base for each fragment can be read Bases are then sorted easily since electrophoresis was used (sequences the DNA)
Application of DNA Technology
Gene Therapy Offers potential cures for individuals with inherited diseases Intended for diseases in which a given gene is mutated or inactive, which gives rise to pathology
DNA AND Biotechnology - detailed lecture notes
Course: Biochemistry/Lab (CHEM 3650)
University: Nova Southeastern University
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