DNA Structure and Function

Learning Outcomes

  • Describe the structure and function of DNA and explain the process by which it encodes for proteins
  • Explain how molecular modeling is used to study the structure and function of DNA as well as other biomolecules.
  • Differentiate between eukaryotic and prokaryotic chromosomal structure and explain how this difference impacts gene regulation in the two cell types
  • Differentiate between bacterial cultures grown in liquid and solid media and explain how to prepare each media type using sterile technique
  • Discuss the characteristics of viruses and their importance in genetic engineering
  • Explain the fundamental process of genetic engineering and give examples of the following applications: recombinant DNA technology, site-specific mutagenesis, and gene therapy
  • Describe the process of gel electrophoresis and explain how the characteristics of molecules affect their migration through a gel

DNA Structure and Function

  • Manipulation of genetic information is central to most biotechnology R&D.
  • Central Dogma of Biology
    • Genes on DNA are transcribed into mRNA.
    • mRNA is translated into protein.
    • This process is also known as "gene expression."

What is DNA?

  • Deoxyribose Nucleic Acid.
  • It is a macromolecule of heredity.
  • Contains the genetic blueprint for life.

DNA's Shape and Storage

  • In one cell, DNA is 2 meters long.
  • If all the DNA in one human body were lined up, it would be 100 trillion meters long.
  • DNA is stored in supercoils called chromosomes.

Chromosomes

  • Supercoils of DNA and proteins.
  • DNA coils around proteins, and these coils are further coiled to form supercoils.
  • Note: The protein that supercoils DNA is a HISTONE.

Chromosome Definition

  • A supercoil of DNA and proteins.

Chromosome Location

  • Chromosomes are located in the nucleus of the cell.
  • DNA is present in every body cell.

CSI Application

  • CSI investigators can use a suspect's blood to convict him of a crime because DNA is present in every single cell.
  • Blood can be compared to other cells left behind, such as saliva or hair.

DNA Structure

  • Two chains of nucleotides twisted into a helix, connected to each other in the center through hydrogen bonds (H-bonds).
  • Each nucleotide contains a sugar molecule, a phosphate group, and a nitrogenous base.

Nucleotides

  • Monomer unit of nucleic acids (DNA/RNA).
  • Composed of 3 parts:
    • A Sugar (5-Carbons, called a pentose):
      • Ribose is in RNA.
      • Deoxyribose is in DNA.
    • A Nitrogenous Base:
      • CTAG in DNA.
      • GUAC in RNA.
    • A Phosphate Group (same in all nucleotides).

DNA Structure: Nitrogenous Bases

  • Nucleotides from each strand bond to each other in the center through H-bonds.
  • Adenine bonds to thymine, and guanine bonds to cytosine.

DNA Structure: Antiparallel Strands

  • The nucleotides in one chain of the helix face one direction, while those in the other strand face the other direction which is called “antiparallel.”
  • The H-bonds holding the antiparallel strands together are rather weak.
  • The two strands of DNA separate easily in high temperatures or in the presence of certain enzymes.
  • This allows for DNA replication and mRNA transcription for protein synthesis.
  • The phosphate and ribose that make up the backbone are held by strong covalent bonds.

Nitrogenous Bases

  • Adenine:
    • 1 of 4 nitrogen bases in DNA.
    • Paired with thymine (weak hydrogen bond).
    • Purine (2 carbon rings).
  • Thymine:
    • 1 of 4 nitrogen bases in DNA.
    • Paired with adenine (weak hydrogen bond).
    • Pyrimidine (1 carbon ring).
  • Guanine:
    • 1 of 4 nitrogen bases in DNA.
    • Paired with cytosine (weak hydrogen bond).
    • Purine (2 carbon rings).
  • Cytosine:
    • 1 of 4 nitrogen bases in DNA.
    • Paired with guanine (weak hydrogen bond).
    • Pyrimidine (1 carbon ring).

Base-Pairing Rules

  • A purine always pairs with a pyrimidine.
  • Therefore:
    • Adenine and thymine are always paired.
    • Guanine and cytosine are always paired.
  • Another way of saying this is that Adenine is complimentary to Thymine and Guanine is complimentary to cytosine.

Complementary Base Pairs

  • In DNA, A pairs with T; C pairs with G.
  • In RNA, A pairs with U; C pairs with G.
  • Give me the compliment of: AAAACGGGGTTTTAAAA
  • IN DNA REPLICATION YOU FORM A "COMPLIMENTARY STRAND"

Discoverers of DNA Structure

  • James Watson & Francis Crick discovered the structure of DNA.
  • They were awarded the Nobel Prize in Physiology and Medicine in 1962.
  • Rosalind Franklin also contributed to the discovery.

Similarities in DNA Molecules Among Organisms

  • All DNA molecules are composed of four nucleic monomers:
    • Adenosine deoxynucleotide (A)
    • Cytosine deoxynucleotide (C)
    • Guanosine deoxynucleotide (G)
    • Thymine deoxynucleotide (T)
  • Virtually all DNA molecules form a double helix.
  • The amount of adenine equals the amount of thymine.
  • The amount of guanine equals the amount of cytosine.
  • Nucleotides in each strand are oriented in the opposite direction of the other strand.
  • Nitrogenous bases.
  • DNA undergoes semiconservative replication.

DNA Replication

  • DNA replicates in a semiconservative fashion in which one strand unzips and each side is copied.
  • It is considered semiconservative since one copy of each parent strand is conserved in the next generation of DNA molecules.

Variations in DNA Molecules

  • The number of DNA strands in the cells of an organism.
  • The length in the base pairs of the DNA strands.
  • The number and type of genes and noncoding regions.
  • The shape of the DNA strands.
  • A single, circular DNA molecule is found in bacteria cells. ~40,000X

Section 4.1 Vocabulary

  • Chromatin – nuclear DNA and proteins
  • Gene – a section of DNA on a chromosome that contains the genetic code of a protein
  • Nitrogenous base – an important component of nucleic acids (DNA and RNA), composed of one of two nitrogen-containing rings; forms the critical hydrogen bonds between opposing strands of a double helix
  • Base pair – the two nitrogenous bases that are connected by a hydrogen bond; for example, an adenosine bonded to a thymine or a guanine bonded to a cytosine
  • Phosphodiester bond – a bond that is responsible for polymerization of nucleic acids by linking sugars and phosphates of adjacent nucleotides
  • Hydrogen bond – a type of weak bond that involved the “sandwiching” of a hydrogen atom between two fluorine, nitrogen, or oxygen atoms; especially important in the structure of nucleic acids and proteins
  • Pyrimidine – a nitrogenous base composed of a single carbon ring; a component of DNA nucleotides
  • Purine – a nitrogenous base composed of a double carbon ring; a component of DNA nucleotides
  • Antiparallel – a reference to the observation that strands on DNA double helix have their nucleotides oriented in the opposite direction to one another
  • Semiconservative replication – a form of replication in which each original strand of DNA acts as a template, or model, for building a new side; in this model one of each new copy goes into a newly forming daughter cell during cell division

Section 4.1 Review Questions

  • Describe the relationship between genes, mRNA, and proteins.
  • Name the four nitrogen-containing bases found in DNA molecules and identify how they create a base pair.
  • The strands on a DNA molecule are said to be “antiparallel.” What does antiparallel mean?
  • During cell division, DNA molecules are replicated in a semiconservative manner. What happens to the original DNA molecule during semiconservative replication?

Sources of DNA

  • In nature, DNA is made in cells.
  • For sources of DNA, use cells in nature or grow cultures of cells in the lab.

Prokaryotic DNA

  • Bacterial Operon
    • An operon contains the controlling elements that turn genetic expression ON and OFF.

R-Plasmids and Transformation

  • Bacteria can transfer plasmids, thus genetic information between themselves.
  • They evolve and thus we call this “transformed”.
  • Scientist have learned how to transfer genes of interest.

Bacterial Cell Culture

  • To grow bacteria cells in the laboratory, a scientist must provide an environment, or medium, that the cells “like.”
  • Some bacteria grow well in a liquid medium (broth).
  • Some bacteria prefer a solid medium, called agar.
  • Some will grow well on or in either.

Eukaryotic DNA

  • Eukaryotic genes have a promoter to which RNA polymerase binds, but they do not have an operator region.
  • Transcription factors may bind at enhancer regions and increase gene expression.

Mammalian Cell Culture

  • Growing mammalian cells in culture is more challenging than growing bacterial cells – more nutrients, etc.
  • Mammalian cells are grown in a broth culture

Introns vs. Exons

  • Gene (DNA): Exon 1, Exon 2, Exon 3, Exon 4, Intron 1, Intron 2, Intron 3
  • Transcription by RNA polymerase
  • Pre-mRNA: Exon 1, Exon 2, Exon 3, Exon 4, Intron 1, Intron 2, Intron 3
  • Splicing (Spliceosome)
  • mRNA: Exon 1, Exon 2, Exon 3, Exon 4
  • Ribosome translates mRNA to protein

Viral DNA

  • Viruses do not have cellular structure.
  • Viruses are tiny, from 25 to 250 nm.
  • They are collections of protein and nucleic acid molecules (DNA or RNA) and become active once they are within a suitable cell.
  • Viruses are classified according to the type of cell they attack:
    • Bacterial (bacteriophages)
    • Plant
    • Animal

Viruses and Gene Therapy

  • Like bacterial plasmid DNA, viral DNA is often used as vectors for genes of interest.
  • Recombinant virus technology=gene therapy.
  • By adding corrective genes (i.e. Therapy genes) into cells with defective genes.
  • I.e. For Treating diabetes, cystic fibrosis, melanoma.

Section 4.2 Vocabulary

  • Medium – a suspension or gel that provides the nutrients (salts, sugars, growth factors, etc.) and the environment needed for cells to survive; plural is media
  • Lysis – the breakdown or rupture of cells
  • R plasmid – a type of plasmid that contains a gene for antibiotic resistance
  • Transformed – the cells that have taken foreign DNA and started expressing the genes on the newly acquired DNA
  • Vector – a piece of DNA that carries one or more genes into a cell; usually circular as in plasmid vectors
  • Operon – a section of prokaryotic DNA consisting of one or more genes and their controlling elements
  • RNA polymerase – an enzyme that catalyzes the synthesis of complementary RNA strands from a given DNA strand
  • Promoter – the region at the beginning of a gene where RNA polymerase binds; the promoter “promotes” the recruitment of RNA polymerase and other factors required for transcription
  • Operator – a region on an operon that can either turn on or off expression of a set of genes depending on the binding of a regulatory molecule
  • Beta-galactosidase – an enzyme that catalyzes the conversion of lactose into monosaccharides
  • Agar – a solid media used for growing bacteria, fungi, plant, or other cells
  • Broth – a liquid media used for growing cells
  • Media preparation – the process of combining and sterilizing ingredients (salts, sugars, growth factors, pH indicators, etc.) of a particular medium
  • Autoclave – an instrument that creates high temperature and high pressure to sterilize equipment and media
  • Enhancer – a section of DNA that increases the expression of a gene
  • Silencer – a section of DNA that decreases the expression of a gene
  • Transcription factors – molecules that work to either turn on or off the transcription eukaryotic genes
  • Intron – the region on a gene that is transcribed into an mRNA molecule but not expressed in a protein
  • Exon – the region of a gene that directly codes for a protein; it is the region of the gene that is expressed
  • Histones – the nuclear proteins that bind to chromosomal DNA and condense it into highly packed coils
  • Nonpathogenic – not known to cause disease
  • Bacteriophages – the viruses that infect bacteria
  • Gene therapy – the process of treating a disease or disorder by replacing a dysfunctional gene with a functional one

Section 4.2 Review Questions

  • Plasmids are very important pieces of DNA. How do they differ from chromosomal DNA molecules?
  • Bacteria cell DNA is divided into operons. Describe an operon using the terms promoter, operator, and structural gene.
  • Describe the human genome by discussing the number and types of chromosomes, genes, and nucleotides.
  • What is gene therapy? Cite an examples of how it can be used.

Isolating and Manipulating DNA

  • “Genetic engineering” is used to describe virtually all directed modifications of the DNA code of an organism.
  • Scientists attempt to alter the genetic code to alter protein production.
    1. Identify a target molecule(s): i.e. insulin
    2. Isolate the instructions (DNA sequence/genes) for the production of the molecule(s): isolate it form humans
    3. Manipulate the DNA instructions: human insulin is pasted into a plasmid and inserted into E. coli cells
    4. Harvesting of the molecule or product, testing it, and marketing it: Humalog by Eli Lilly and Company

Genetic Engineering Techniques

  • Recombinant DNA Technology (rDNA technology):
    • Methods to create new DNA molecules by piecing together different DNA molecules.
    • I.e. R-insulin, rh insulin
  • Site-Specific Mutagenesis:
    • A technique that involves changing the genetic code of an organism (mutagenesis) in certain sections (site-specific) on a particular DNA code.
    • Ie using Chemicals, radiation, or viruses (e. UV light causes cell growth).
    • Tide laundry detergent contains rSubtilisin protease that removes blood gravy and other protein stain
  • Gene Therapy:
    • Process of correcting faulty DNA codes that cause genetic diseases and disorders (Parkinson's)

Section 4.3 Review Questions

  1. Genetic engineering by any method requires certain steps. Put the following steps in the correct order:
    • isolation of the instructions (DNA sequence/genes)
    • harvest of the molecule or product; then marketing
    • manipulation of the DNA instructions
    • identification of the molecule to be produced
  2. What “naming” designation is used with recombinant products made through genetic engineering?
  3. What is the smallest change in a DNA molecule that can occur after site-specific mutagenesis? What effect can this change have?
  4. What gene has been the target of CF gene therapy? What does this gene normally do?

Using Gel Electrophoresis to Study Gene Molecules

  • Components of Gel Electrophoresis:
    • Agarose dissolved in electrophoresis buffer
    • Electrophoresis gel box
    • Power supply
    • Visualization system
  • Gel electrophoresis uses electricity to separate molecules, based on the size, shape, and charge, in a gel slab
  • Hot agarose solution is poured into a gel tray.
  • A comb is added to form sample wells as the agarose cools and solidifies.
  • Wells are formed at one end so that sample molecules can move across the gel and separate based on their characteristics.

Gel Box with Gel and Buffer

  • For the gel box to conduct electricity and establish an electric field with a positive end (red wire) and a negative end (black wire), the solution in the gel box must contain ions.
  • Sodium chloride (NaCl) solution can be used, but other salts, such as TRIS or lithium, dissolved in water (called a “running buffer”), are better for conducting electricity.
  • Agarose gel electrophoresis is best used when separating pieces of DNA no smaller than 50 bp and no larger than 25,000 bp.

Agarose Gel Concentrations

  • Agarose gels are made with concentrations ranging from 0.6% to 3% agarose in buffer.
  • The concentration of a gel is of critical importance.
  • The more agarose molecules in solution, the more strands intertwine to make the “strainer.”
  • If the concentration is too high, larger molecules can’t move through the long, woven agarose molecules.
  • The most common gel used for DNA fragment separation is 0.8% agarose which is good for most plasmid and restriction digestion fragments separate well.
  • “Tighter” gels (2% or 3%) are used to separate smaller molecules, such as PCR products.
  • Proteins and nucleic acids that are smaller than 50 bp are run on polyacrylamide gels in vertical gel boxes.

DNA Gel Stains

  • Nucleic acids are colorless, so technician must “stain” gels to see the bands of separated molecules.
  • There are a few DNA stains to choose from.
  • An ethidium bromide stained gel. Each white band is thousands of DNA molecules of similar size. These bands are PCR products that are 500 to 1000 bp in length.
    • Ethidium bromide (EtBr) is the most common DNA gel stain in most labs and considered the most sensitive to low concentrations of DNA. EtBr glows orange when it is mixed with DNA and exposed to UV.
    • Since EtBr is a suspected mutagen, other stains such as LabSafe®, SYBR Safe, GelRed, GelGreen, or methylene blue have become popular.
    • Each of these stains interacts with the nucleic acid molecules in a way that they can be visualized by either UV or white light. Each has some pros and some cons in its use.

DNA Samples on a Gel

  • Lane 1: DNA sizing standard (Lambda/HindIII)
  • Lane 2: DNA sample about 7000 bp (plasmid)
  • Lane 3: Plasmid restriction digestion
  • Lane 4: Genomic Bacterial DNA
  • Lane 5: mRNA
  • Lanes 6/7: Smears of DNA, assorted size pieces
  • Lane 8: No nucleic acid
  • Lane 9: DNA strands, large, will not “load”
  • Lane 10: No sample
  • This gel represents what DNA samples from eukaryotic and prokaryotic sources might look like on a 0.8% agarose gel.

Section 4.4 Vocabulary

  • Gel electrophoresis – a process that uses electricity to separate charged molecules, such as DNA fragments, RNA, and proteins, on a gel slab
  • Agarose – a carbohydrate from seaweed that is widely used as a medium for horizontal gel electrophoresis
  • Polyacrylamide – a polymer used as a gel material in vertical electrophoresis; used to separate smaller molecules, like proteins and very small pieces of DNA and RNA
  • Ethidium bromide – a DNA stain (indicator); glows orange when it is mixed with DNA and exposed to UV light; abbreviated EtBr
  • Methylene blue – a staining dye/indicator that interacts with nucleic acid molecules and proteins, turning them to a very dark blue color
  • High through-put screening – the process of examining hundreds or thousands of samples for a particular activity

Section 4.4 Review Questions

  1. Agarose gels can be used to study what size of DNA fragments?
  2. If agarose gel material is labeled 1%, what does the 1% refer to?
  3. What causes molecules to be separated on an agarose gel?
  4. Name two common DNA stains that are used to visualize DNA on agarose gels.