DNA Structure & Function Every cell within an organism has the same DNA (genome) – excluding sex cells. BUT the entire genome is variable from organism to organism. Although there are differences in the genomes of different organisms – all DNA shares many characteristics. Similarities in DNA among organisms: 1. DNA molecules are composed of 4 nitrogenous bases within the nucleotides – Adenine (A), Thymine (T), Cytosine (C), Guanine (G). 2. All DNA molecules form a double helix of repeating nucleotides. Nucleotides form phosphodiester bonds between the sugars and phosphates of adjacent nucleotides. Hydrogen bonds are found between complementary nitrogen bases. A-T, C-G 3. Complementary base pairs equal in amounts. A=T, C=G 4. The nucleotides in each strand are oriented in the opposite direction – antiparallel. This arrangement allows DNA to be “read” in one direction – 5’ to 3’. 5. Nitrogenous bases are stacked .34 nanometers (nm) apart with 10 nitrogen bases per complete turn of the helix. This stacking ensures the strands are consistently parallel. This uniform shape ensures that enzymes and regulatory molecules can recognize the DNA molecule and allows for coiling and packing of the DNA. 6. DNA undergoes semiconservative replication – making two daughter strands from a single parent strand. During replication, a strand unzips and each single strand acts as a template for building a new side. At the end of replication, two identical DNA strands have been produced. Variations in DNA molecules: 1. Number of DNA strands in the cells of an organism (# of chromosomes) 2. Length of base pairs of DNA strands 3. Number and type of genes (nucleotides that code for a protein) and noncoding regions 4. The shape of the DNA strands (circular or linear)

DNA in Organisms For sources of DNA, scientist can grow cultures of cells in the lab. Growth of cells occurs on or in a medium (source of nutrients). Cells from cell cultures can be collected and broken open – process called lysis. Lysed cells release DNA molecules that can be isolated form the other cell molecules. The packing of the DNA and location within the cell are different – knowing this difference will help the isolation process. *Adapted from “Biotechnology: Science for the New Millennium” by Ellyn Daugherty.

Prokaryotic DNA  Bacterial cells are prokaryotic – does NOT contain a nucleus or membrane-bound organelles. DNA is floating in the cytoplasm, but is usually attached to one spot to the plasma membrane. A bacterium contains only one long, circular DNA molecule – it is usually supercoiled. The DNA contains only several thousand genes. Example: E. coli genome codes for RNA and proteins with very little unused DNA.  Some bacteria contain extra rings of DNA floating in the cytoplasm – these are called plasmids. A plasmid contains only a few genes (5 to 10). These genes usually code for proteins that offer some additional characteristic that may be needed under extreme conditions. Example: R plasmids contain antibiotic resistance genes.  Bacteria can transfer plasmids – transferring genetic information between themselves. Transferring plasmids may give bacteria a way of “evolving” by gaining new characteristics.  Scientists have learned how to use plasmids to transfer “genes of interest” into cells. Cells can take up foreign DNA and start expressing the genes – these bacteria are transformed.  Different bacteria have different plasmids – some have different types and some have none.  Plasmids are often used as recombinant DNA (rDNA) vectors to transform cells. Foreign DNA fragments (genes) are cut and pasted into a plasmid vector. The recombinant plasmid is introduced into a cell. The cell reads the gene on the plasmid and starts synthesizing the protein coded on the “foreign gene”.  Restriction enzymes evolved as a defense mechanism to protect bacteria from invading viruses. Bacteria produce restriction enzymes to cut viral DNA which destroys the virus. Restriction enzymes splice (cut DNA) and are used to splice DNA in preparation for gel electrophoresis. Examples of restriction enzymes: EcoRI, HindIII and Pstl. Gene Expression in Prokaryotes  Gene expression (how genes are turned off and on) is simple with only a few controls. Bacteria can have several operons – section of prokaryotic DNA consisting of one or more genes and their controlling elements. In the middle of the operon is a structural gene that actually codes for one or more mRNA molecules – this will be translated to form a functional protein.

*Adapted from “Biotechnology: Science for the New Millennium” by Ellyn Daugherty.

 For gene expression to occur in prokaryotes, an enzyme that synthesizes mRNA – RNA polymerase – must attach to a segment of DNA at a promoter region of the operon. This turns “ON” the gene. The RNA polymerase works down the DNA strand to a structural gene – it then builds a mRNA molecules using the DNA as a template. The synthesized mRNA leaves the nucleus and is decoded into a polypeptide chain at the ribosome.  The operator (region prior to the structural gene) can ‘turn off’ the operon. If a regulatory molecules attaches at the operator, the operon is turned off because the RNA polymerase is blocked from continuing down the strand to the gene – no protein is produced. Blocking and unblocking the operator is a way bacterial cells make only certain proteins at certain times.  Genetic engineers utilize the promoter and operator regions to turn on and off the production of certain genes. Eukaryotic DNA  Eukaryotic DNA (protist, fungi, plant and animal cells) is packed into chromosomes, regulated and expressed differently from that of bacteria. Eukaryotes have several chromosomes per cell and each chromosome is a single, linear molecule of DNA coiled around proteins (histones). Each single DNA molecule may contain several million nucleotides and many genes.  The total amount of DNA per cell is not directly related to an organism’s complexity. Much of eukaryotic DNA is noncoding – meaning it does not transcribe into genes. Much of the DNA in higher organisms is spacer DNA within and between genes – widely spaced genes provide an evolutionary advantage. Genes that are far apart are often involved in recombination – shuffles forms of a gene from one chromosome to another. This leads to new combinations being sent to sex cells – results in increased diversity in the next generation. Gene Expression in Eukaryotes – Central Dogma  Eukaryotic cells lack operators in their DNA. Eukaryotic genes contain a “promoter region” – where an RNA polymerase molecule binds. The RNA polymerase moves along the DNA molecule until it finds the structural gene(s). At the structural gene, the RNA polymerase builds a complementary mRNA transcript from one side of the DNA strand. The enzyme transcribes the entire gene until it reaches a termination sequence.  Eukaryote genes are usually turned “on” and expressed at a very low level. Expression is increased or decreased when molecules interact with enhancer or silencer regions on the DNA. The enhancer or silencer regions may be within a gene or elsewhere on the chromosome. The molecules that bind at enhancer or silencer regions are called transcription factors (TF).

*Adapted from “Biotechnology: Science for the New Millennium” by Ellyn Daugherty.

 mRNA transcripts are modified before translation. Structural genes are composed of intron and exon sections. Exons are DNA sections that contain the protein code – they are expressed. The introns are usually spacer DNA. A polymerase molecule attaches at the promoter and moves down an entire structural gene including the intron sections. Upon completion, the introns (noncoding regions) are removed so that only the exons (coding regions) remain on the mRNA molecule. The mRNA molecule is decoded into a protein at a ribosome.  Eukaryotic genes are regulated by the way the chromosomes are coiled. Chromosomes in higher organisms are highly coiled around structural proteins (histones). The histone-DNA complex coils on itself again and again, which conceals genes. When genes are buried this way, RNA polymerase cannot transcribe them into mRNA. The gene has been turned “OFF”. DNA has to uncoil all the way to expose the DNA helix to be transcribed and translated to protein.

Viral DNA  Nonpathogenic viruses or virus particles are often used in biotechnology research as vectors to carry DNA between cells. Viruses are nonliving and do NOT have cellular structure. Viruses are composed of proteins and nucleic acid molecules that become active once they are within a suitable host cell.  Virus size ranges from 25 to 250 nm. Viruses are classified into main categories based on the type of cell they attack – bacterial (bacteriophages), plant and animal.  Viruses have a thick protein coat surrounding a nucleic acid core of either DNA or RNA. Viral Replication  Within a host cell, the viral nucleic acid is released – the viral genes are read by the host cell’s enzymes, decoded into viral mRNA and translated into viral proteins. New viruses assemble and release from the host cells to infect other cells – these viruses are lytic viruses.  Some viruses incorporate their DNA into the host chromosomes when released in the host cell – these viruses are lysogenic viruses. Biotechnology Uses of Viruses The viral particle structure is important in trying to control viruses. Therapies focus on the ability of the human immune system to recognize viral surface proteins. Viral vaccines recognize specific viral surface proteins and target them for attack. Some biotech companies are developing protease inhibitors – they destroy proteases made by a virus in their attempts to take over host cells. Viral DNA or RNA molecules are short and easy to manipulate – viral DNA is used as vectors. Viral DNA molecules may be cut open to insert genes of interest. When sealed, the viral DNA becomes recombinant molecules that can be inserted into host cells or the virus itself. If the recombinant DNA is inserted back into virus, the virus can insert the rDNA into an appropriate host cell. Recombinant virus technology is one technique used in gene therapy. Viruses can insert corrective genes into cells that contain defective genes. Example: Many companies are trying gene therapy to treat diabetes, cystic fibrosis, other genetic disorders or cancer.

*Adapted from “Biotechnology: Science for the New Millennium” by Ellyn Daugherty.

Manipulating DNA Modifications of DNA can range from a single nitrogen base change to cutting entire genes and inserting new ones. Changes in DNA may alter the production of proteins – new proteins may be created or protein production can stop. Genetic Engineering describes the production of rDNA molecules (pieces of DNA cut and pasted together – recombinant DNA) and the insertion of rDNA into cells. The process of genetic engineering requires the following steps: 1. Identification of the molecules which could be produced more easily or economically through genetic engineering – example: insulin for diabetics. 2. Isolation of DNA for the production of the molecule – example: insulin gene. 3. Manipulation of DNA by changing them inside the cell or by putting DNA into another cell that can produce the molecule more easily, in larger amounts or less expensively – example: insulin gene is pasted into a plasmid and inserted into E.coli cells. 4. Harvesting the product, testing it and marketing it to the public – harvesting insulin from tanks, testing and formulated for distribution. Site-Specific Mutagenesis The process of inducing changes (mutagenesis) in certain sections (site-specific) of a particular DNA code – the DNA changes are usually accomplished through the use of chemicals, radiation or viruses. Bacteria, plant, fungi or animal cell cultures may be treated with mutagens – for example: exposure of bacteria colonies to different amounts of UV light radiation will increase cell growth, new pigment production or cell death. Mutagenic agents have various effects – may cause substitutions (replacement of one base with another) OR additions/deletions to large sections of DNA. These mutations may cause a change in protein production. Site-specific mutagenesis is “directed” to make certain changes to alter protein structure that will translate into an improved function. Gene Therapy The process of correcting or modifying DNA codes that causes genetic diseases or disorders. Two common ways to introduce new genes into defective cells: (1) Use a virus to carry a new or normal gene into target cells, (2) To package a “good” gene in a synthetic lipid envelope (liposome) and use the envelope to bring the gene into a cell.  Example: Cystic Fibrosis causes a buildup of mucus – predisposes a patient to lung infections. In 2002, a modified cold virus was used to transfer a normal copy of the cystic fibrosis transmembrane conductance regulator gene (CFTR) to cells lining the nose. The CFTR regulates the flow of Chloride ions into cells and is defective in CF patients. Restriction Enzymes Restriction enzymes (endonucleases) cut or digest DNA from any source at a specific site – enzymes hydrolyze the sugar phosphate bond between two specific nucleotides in the restriction site on each DNA strand breaking the DNA in a predictable location. Some enzymes cut across both DNA strands leaving “blunt” ends with no unpaired bases and some make staggered cuts in the DNA creating “sticky” ends with unpaired bases. Enzymes can cut in more than one location at specific sites to create multiple DNA fragments. Restriction sites are usually 4 to 6 bases long. Once restriction enzymes cut DNA into fragments, the fragments can be moved and joined together using ligase (enzyme). Ligases reform the phosphate bonds that were broken by the restriction enzymes. *Adapted from “Biotechnology: Science for the New Millennium” by Ellyn Daugherty.

RFLP & SOUTHERN BLOTTING In 1985, Alec Jeffreys invented DNA profiling which is based on variances in the DNA sequences among individuals. Genetic variations among individuals can include mutations in restriction sites – these genetic differences are called restriction fragment length polymorphisms (RFLP). RFLP analysis generates a banding pattern unique for each individual – a DNA fingerprint.  RFLP is a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after restriction digestion.  When performing RFLP analysis on genomic DNA, the DNA is digested with restriction enzymes and the fragments generated are analyzed by gel electrophoresis. There are thousands of restriction sites on DNA and the fragments smear. To create distinct bands and detect RFLPs, only a select number of DNA fragments are visualized using Southern Blotting – named after Edwin Southern (invented in 1975).  In Southern Blotting, genomic DNA is resolved on an agarose gel, denatured so that it is singlestranded and transferred onto a solid membrane of nylon or nitrocellulose. DNA bands are trapped by the membrane and form a replica of the gel. A probe (short piece of complimentary DNA) is incubated with the membrane – the probe is chemically modified with either a radioactive or fluorescent label so it can be detected. The location of the tagged probe and the distinct banding pattern of DNA are visualized using photographic film. Southern blotting requires a large amount of DNA and is time-consuming – there is no risk of contamination and results are indisputable.  RFLP probes are frequently used in genome mapping and in variation analysis (genotyping, forensics, paternity tests, heredity disease diagnosis, etc.)

POLYMERASE CHAIN REACTION In 1983, Kary Mullis invented polymerase chain reaction (PCR) – replication of a specific sequence of DNA to create billions of copies. Single strands of DNA called primers target a sequence and bind (anneal) each end of the target sequence. Primers provide the specificity of PCR, selecting the region to be amplified. PCR occurs in three stages: 1. Denaturation – denature the template DNA by heating the reaction to 94oC. The high temperature causes DNA double helix to separate by breaking hydrogen bonds between base pairs, resulting in a single-stranded DNA. 2. Annealing – Primers anneal to the target sequence. The reaction is cooled to allow hydrogen bonds to form between the primers and the single-stranded template DNA – this temperature is known as the annealing temperature. The optimal annealing temperature is usually 50-60oC – this temperature prevents DNA from reforming into a double-strand. 3. Extension – DNA polymerase binds to regions of double-stranded DNA created by the binding of the primer and the template DNA. The DNA polymerase reads the template strand in the 3’ to 5’ direction and adds bases in the 5’ to 3’. Cycle is repeated 25-40 times to amplify the target sequence.  A thermal cycler is used to quickly change temperatures during the polymerase chain reaction.  A quantitative or real-time PCR allows for the quantification of initial DNA in a sample – amount is measured as each cycle is completed and is used to deduce the amount of input DNA.

*Adapted from “Biotechnology: Science for the New Millennium” by Ellyn Daugherty.

GEL ELECTROPHORESIS This process uses electricity to separate molecules (DNA, RNA and proteins) in a gel slab – molecules separate based on their size, shape and charge. The process of gel electrophoresis requires the following steps: 1. Make the gel – agarose (carbohydrate derived from seaweed) is dissolved in a boiling buffer solution. The solution is poured into trays – a comb is placed in the liquid gel to form the wells.  Agarose gels are commonly made with concentrations ranging from .6% to 3% agarose in buffer. The concentration of gel is important – more agarose concentration makes it difficult for larger molecules to move.  DNA gels are .8% agarose. Tighter gels (2% or 3%) are used to separate smaller molecules. Agarose gels with high concentration are hard to prepare, so acrylamide gels are mostly used. 2. The solidified gel is placed in a gel box and covered with buffer solution. The gel box has electrodes at each end – electric current runs in the gel box.  Most buffers contain TRIS – a buffering salt that stabilizes pH, maintains shape of molecules being analyzed and conducts electricity. 3. A sample is loaded into the wells – when power is turned on, the electric field is established and charged molecules move. If molecules have a negative charge, they move toward the positive end; if molecules have a positive charge, they move toward the negative end.  DNA and most molecules are colorless – samples are mixed with loading dye to make the sample easy to use.  To determine the size of unknown DNA fragments, use a DNA size standard of known fragment sizes. DNA size standards are called standards, ladders, rulers or markers. 4. The gel acts as a molecular strainer – separates fragments based on size, shape and charge.  Two common gels are agarose and polyacrylamide: Agarose gels are used in horizontal gel boxes to study medium to large pieces of DNA – Polyacrylamide gels (PAGE) are used to separate smaller molecules such as proteins and small pieces of DNA/RNA. Molecule DNA RNA Proteins

Carbohydrates Lipids

Charge Negative Negative Positive Negative Neutral Most are neutral. Most are neutral.

Size 500-25,000 bp < 1,000 bp 1000-350,000 Da 1000-350,000 Da 1000-350,000 Da Variable Variable

Behavior Moves to (+) pole; small molecules move faster Moves to (+) pole; small molecules move faster Moves to (-) pole; small molecules move faster Moves to (+) pole; small molecules move faster No net movement to either pole No net movement to either pole No net movement to either pole

5. Once a gel is prepared, laded with a sample, it is ready to run. The voltage is set at about 110 volts (V). Depending on the buffer concentration, 110V produces current of about 35-80 mAmps. As current moves through the gel, the molecules move into the gel at different rates. 6. Since nucleic acids are colorless, the gels must be stained to see the bands of separated molecules. Two common stains: (1) Ethidium Bromide (EtBr) glows orange when mixed with DNA and exposed to UV light. *EtBr is a suspected mutagen. (2) Methylene Blue will bond with nucleic acid molecules turning them blue.  In many labs, hundreds of samples are run on agarose gels at the same time – this is high through-put screening.

*Adapted from “Biotechnology: Science for the New Millennium” by Ellyn Daugherty.

BIOINFORMATICS – Bioinformatics is the use of technology and computer science to perform biological research -essential to help sort and evaluate data sets too large to be processed manually. Technicians apply computer processing to data such as DNA sequences, protein sequences and protein structures to generate new information. Bioinformaticians use databases that contain data derived by scientists over many years.  One of the largest databases is GenBank that host billions of DNA and protein sequences. GenBank is operated by the National Center for Biotechnology Information and is funded by the U.S. National Institutes of Health (NIH).  Scientists also use GenBank to find sequences that are similar to those they are investigating by using bioinformatics tools such as the Basic Local Alignment Search Tool (BLAST). BLAST companies input DNA or protein sequences with sequences in GenBank and finds the sequences that are most closely match the input sequence. This allows researchers to match unknown sequences to known genes or proteins, to verify sequences and to find differences in sequences from different species.  The tertiary and quarternary structures of proteins and their interactions with binding partners are predicted using a sub-discipline of bioinformatics called protein modeling. Protein modeling methods help explain how proteins function. The 3-D structures of proteins are determined by analyzing x-ray diffraction and nuclear magnetic resonance (NMR) and stored in the Protein Data Bank (PDB).  ClustalW can be used to align amino acid sequences of proteins. Bioinformatics are also used to map genomes and generate phylogenetic trees.

HS-AB-2: Describe how characteristics of living organisms are integrated with advanced biotechnology techniques to lead to discovery or production. 2.2 Demonstrate how DNA structure and function may be exploited in genetic engineering to produce specific genetic constructs. 2.3 Engineer nucleic acids through selecting, excising, ligating and cloning of plasmid or viral vectors for development of molecular delivery systems. 2.4 Simulate enzymatic replication of nucleic acids utilizing real-time or traditional PCR including primer design. 2.5 Isolate and prepare DNA samples for sequencing. 2.6 Manage and analyze DNA sequence data using bioinformatics tools (e.g. Genbank and BLAST). 2.7 Relate principles of macromolecule structure, physical chemistry and composition to strategies for isolating, analyzing and characterizing protein and DNA. 2.10 Apply the principles of electricity and ionization to successfully migrate charged molecules in ionic buffering systems. *Adapted from “Biotechnology: Science for the New Millennium” by Ellyn Daugherty.