Chapter 4: Introduction to Studying DNA
To grow bacteria cells you must provide a medium that the cells
"like". Some bacteria grow well in liquid medium (broth), solid medium (agar), some will grown on or in either
components of gel electrophoresis
- agarose dissolved in electrophoresis buffer (NOT water) - electrophoresis gel box -power supply -visualization system
Vriuses are classified according to the type of cell they attack:
- bacterial (bacteriophages) - plant - animal
What are some 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
Agarose gels are made with concentration from
0.6% to 3% agarose in buffer
The most common gel used for DNA fragment seperation is _______ agarose which is good for most plasmid and restriction digestion fragments seperate well
0.8%
All DNA molecules are composed of four nucleic monomers
1. Adenosine deoxynucleotide 2. Cytsoine deoxynucleotide 3. Guanosine deoxynucleotide 4. Thymine deoxynucleotide
steps of genetic engineering
1. Identify a target molecule 2. Isolate the instructions (DNA sequence/genes) for the production of the molecule(s) 3. Manipulate the DNA instructions 4. Harvesting of the molecule or product, testing it, and marketing it
steps of gel electrophoresis
1. hot agorase solution is poured into a gel tray 2. a comb is added to form sample wells as the agarose cools and soldifies 3. Wells are formed at one end so that sample molecules can move across the gel and seperate based on their characteristic
Describe the human genome by discussing the number and types of chromosomes, genes, and nucleotide
46 chromosomes, 23 pairs> 20,000 genes in human genome > 3 billion base pairs
agarose gel electrophoresis seperate pieces of DNA no smaller than
50 bp and no larger than 25,000 bp
Polyacrylamide
A polymer used as a gel material in vertical electrophoresis; used to separate smaller molecules, like proteins or very small pieces of DNA or RNA
RNA polymerase
An enzyme that catalyzes the synthesis of an RNA strand from a DNA template
What gene has been the target of CF gene therapy? What does this gene normally do?
CFTR--> regulated the flow Cl- into epithelial cells in respiratory and digestive system
What is the most common DNA gel stain in most labs and considered the most sensitive to low concentrations of DNA
Ethidium bromide (EtBr)
What must be done for gel box to conduct electricity?
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
What is the center of most biotech R&D?
Manipulation of genetic info
"Tighter" gels (2% or 3%) are used to seperate smaller molecules such as
PCR products
What are solution good for conducting electricity?
Sodium chloride (NaCl) can be used but other other salts such as TRIS or lithium dissolved in water (called a "running buffer") are better for conducting electricity
Why is concentration of a gel of critical importance?
The more agarose molecules in solution, the more strands intertwine to make the "stainer" . If the concentration is too high, larger molecules can't move through the long, woven agarose molecules
DNA: Nitrogenous bases
The nucleotide from each strand bond to each other in the center through H-bonds. The nucleotide containing adenine bonds to thymine and guanine bonds to cytosine
DNA Structure: Antiparallel Strands
The nucleotide in one chain of the helix faces one direction, while those in the other strand face the other direction. Called "antiparallel". The H-bonds holding the antiparallel strands together are rather weak; therefore, the two strands of DNA separate easily in high temps or in the presence of certain enzymes. This allows for DNA replication and mRNA transcription for protein synthesis.
DNA samples on a gel
This gel represents what DNA samples from eukaryotic and prokaryotic sources might look like on a .8% agarose gel.
3D Model of DNA
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 mlecule, a phosphate group, and a nitrogenous base
Each of these stains interacts with the nucleic acid molecules in a way they can be visualized by either _____ or _____ _____. Each has some pros and some cons in its used
UV or white light
ethidium bromide
a DNA stain (indicator); glows orange when it is mixed with DNA and exposed to UV light; abbreviated EtBr
Phosophodiester bond
a bond that is responsible for polymerization of nucleic acids by linking sugars and phosphates of adjacent nucleotide
Agarose
a carbohydrate from seaweed that is widely used as a medium for horizontal gel electrophoresis
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
Purine
a nitrogenous base composed of a double carbon ring; a component of DNA nucleotides
Pyrimidine
a nitrogenous base that has a single carbon ring; a component of DNA nucleotides
Vector
a piece of DNA that carries one or more genes into a cell usually circular as in plasmid vectors
Gel electrophoresis
a process that uses electricity to separate charged molecules, such as DNA fragments, RNA, and proteins, on a gel slab
antiparallel
a reference to the observation that strands on DNA double helix have their nucleotides oriented in the opposite direction to one another
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
Gene
a section of DNA on a chromosome that contains the genetic code of a protein
Silencer
a section of DNA that decreases the expression of a gene
Enhancer
a section of DNA that increases the expression of a gene
Operon
a section of prokaryotic DNA consisting of one or more genes and their controlling elements such as operator and promoter
Agar
a solid media used for growing bacteria, fungi, plant, or other cells
methylene blue
a staining dye/indicator that interacts with nucleic acid molecules and proteins, turning them to a very dark blue color
Medium
a suspensions or gel the provides the nutrients (salts, sugars, growth factors, etc.) and the environment needed for cells to survive; plural is media
site-specific mutagenesis
a technique that involves changing the genetic code of an organism (mutagenesis) in certain sections (site-specific). Process of including changes (mutagenesis) in certain sections (sit-specific) on a particular DNA code
R plasmid
a type of plasmid that contains a gene for antibiotic resistance
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
Beta-gelactosidase
an enzyme that catalyzes the conversion of lactose into monosaccharides
Nitrogenous base
an important component of nucleic acids (DNA and RNA), composed of one or two nitrogen-containing rings; forms the critical hydrogen bonds between opposing strands of a double helix
Autoclave
an instrument that creates high temperature and high pressure to sterilize equipment and media
Bacterial operon
an operon contains the controlling elements that turn genetic expression ON and OFF. Prokaryotic cells
Size of virus
are tiny from 25 to 250 nm
Why are other stains such as LabSafe, SYBR Safe, Gelred, Gelgreen, or methylene blue have become popular?
because EtBr is a suspected mutagen
Why is growing mammalian cells more challenging than bacterial cells?
because there are more nutrients, etc.
In what medium is mamallian cells grown in?
broth culture
Viruses do not have a
cellular structure
What is the smallest change in DNA molecule that can occur after site-specific mutagenesis? What effect can this change have?
change or removing a simple nucleotide--> change shape or function of protein
Virtually all DNA molecules form a
double helix
gel electrophoresis uses _____ to separate molecules, based on the size, shape, and charge in a gel slab
electricity
Proteins are produced when
genes on a DNA molecule are transcribed into mRNA, and mRNA is translated into the protein code. This is called "gene expression"
Eukaryotic DNA
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
Genetically 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
Broth
liquid media used for growing cells
Recombinant DNA technology
methods to create new DNA molecules
Transcription factors
molecules that work to either turn on or off the transcription eukaryotic genes
Nonpathogenic
not known to cause cancer
Chromatin
nuclear DNA and proteins
When do viruses become active?
once they are within a suitable cells
How does DNA replicate?
one strand unzips and each side is copied.
Nucleotides in each strand are oriented in the ____ direction of the other strand
opposite
What colors to EtBr glow when mixed with DNA and exposed to UV
orange
Proteins and nucleic acids that are smaller than 50 bp are run on ____ gels in vertical gel boxes
polyacrylamide
Viruses are a collections of
protein and nucleic acid molecules (DNA or RNA)
What "naming" designation is used with recombinant products made through genetic engineering?
r in front of the name
What is gene therapy> Cit an examples of how it can be used
replace faulty genes with functioning gene. Cystic fibrosis
DNA undergoes _______ replication
semiconservative
Plasmids are small and used to
shuttle in DNA into organisms
Why is DNA considered semi-conservative?
since one copy of each parent strand is conserved in the next generation of DNA molecules
Bacteria cell DNA is divided into operons. Describe an operon using the terms promoter, operator, and structural gene
structural gene encodes for a protein promoter binds RNA polymerase operator controls when gene is expressed or not
Lysis
the breakdown or rupture of cells
Transformed
the cells that have taken foreign DNA and started expressing the genes on the newly acquired DNA
Histones
the nuclear proteins that bind to chromosal DNA and condense it into highly packed cells
media preparation
the process of combining and sterilizing ingredients (salts, sugars, growth factors, pH indicators, etc) of a particular medium
High through-put screening
the process of examining hundreds or thousands of samples for a particular activity
Gene therapy
the process of treating a disease or disorder by replacing a dysfunctional gene with a functional one
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
Exon
the region of a gene that directly codes for a protein; it is the region of the gene that is expressed
Intron
the region on a gene that is transcribed into mRNA molecule but not expressed in a protein
Each white band in EtBr stainged gel is ____. What ength?
thousands of DNA molecules of similar size. 500 to 1000 bp in length
Base pair
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
Bacteriophages
viruses that infect bacteria
