Chapter 20: DNA Technology
what is one use of gel electrophoresis?
"DNA fingerprinting" in murder/rape trials
banding pattern
DNA is shocked and travels through the agarose gel, with the heavier pieces staying close to the wells and the light picees migrating the farthest; during gel electrophoresis, very large DNA molecules often yield so many fragments that they appear as a smear instead of distinct bands
what is the most useful procedure in mapping the genome of an organism?
DNA sequencing
pharmaceutical products
DNA technology makes it possible to produce these by transferring the gene for a desired trait into bacteria or yeast (insulin, growth hormone, tissue plasmogen activator (TPA), helps dissolve blood clots and reduce risks of heart attack)
nucleic acid probe
a nucleotide sequence that is complimentary to the desired gene; labeled with a radioactive isotope or a fluorescent tag because under UV light only the colony with the gene of interest will have bonded to the probe and glow
Southern blotting
a technique used to detect and compare specific DNA fragments among individuals; uses single-stranded radioactive DNA probes that are complimentary to a gene or DNA fragment of interest
gel electrophoresis
a technique used to study and compare how genes and nucleotide sequences differ between individuals; separates segments of DNA, RNA, or protein based on their size
audiography
after the DNA is cut with restriction enzymes and run through the agarose gel by gel electrophoresis, the bands are transferred to nitrocellulose paper, which denatures the DNA, separating the strands so a complimentary nucleic acid probe can bond to and illuminate the genes or nucleotide sequences of interest; then paper is laid over the blot, and the radioactive probes expose the film and form an image of the genes of interest
two important genes on plasmids
ampR gene, lac Z gene
agricultural uses
animal husbandry, genetic engineering in plants, nitrogen fixation
in situ hybridization
another technique to find the specific location of a gene or nucleotide sequence on a chromosome; radioactive probes base pair with a complementary sequence of DNA on an intact chromosome, which is mounted on a microscope slide and stained to be observed where the probe has attached
production of proteins of interest in large quantities
bacteria containing the gene for human growth hormone can make large quantities of the hormone to treat stunted growth (insulin also)
why use PCR?
bacterial cells are effective in producing large quantities of a gene, but PCR can make multiple copies of a gene in a much shorter time
problems with introns
bacterial genes do not have introns and bacterial cells lack the RNA splicing machinery to take the introns out of eukaryotic genes
problems with promoters
bacterial promoters are different than eukaryotic promoters; prokaryotic organisms do not recognize the promoter regions of eukaryotic genes and these genes cannot be expressed
techniques used in DNA technology
bacterial transformation, polymerase chain reaction, gel electrophoresis
what are some examples of transgenic organisms?
beef, dairy cattle, sheep, commercial fish, etc. have all been produced by injecting foreign DNA into egg cells; rainbow trout and salmon injected with foreign GH genes can grow in one year the same size it would normally take them 2-3 years to grow
genetically engineered proteins as a pharmaceutical product
block or mimic surface receptors on cell membranes; protein mimic receptors that HIV binds to; cannot enter/infect white blood cellss
how can scientists further avoid eukaryotic/prokaryotic incompatibility?
by using eukaryotic cells as hosts, such as yeast , for gene expression
diagnosis of disease
can use PCR and labeled nucleic acid probes to identify pathogens (amplifies and detects HIV and AIDS in blood and tissue samples); can identify individuals with genetic diseases before the onset of symptoms or birth (hemophilia, PKU, cystic fibrosis, Duschene's muscular dystrophy
heat shocking
causes cell to open pores
what happens once the bacterial colony carrying the gene of interest is identified?
cells are grown in a liquid culture; can easily isolate and make numerous copies of the gene
ampR gene
codes for antibiotic resistance (ampicillin)
lac Z gene
codes for beta galactosidase, the enzyme that breaks down lactose
cDNA
complimentary DNA; DNA with no introns that can be expressed by bacterial cells as long as it is inserted near an active bacterial promoter
nitrogen fixation
conversion of nitrogen gas into nitrogen containing compounds that plans use; carried out by bacteria in the soil or roots of plants like beans and alfalfa
what are some examples of animal husbandry?
cows injected with BGH, increasing milk production and the weight gain in cattle; cellulase; transgenic organisms
DNA sequencing
determines actual nucleotide sequences of genes and chromosomes; once DNA sequences are collected, they are put into computer data banks where scientists can scan sequences for start/stop codons and compare the sequences being studied to sequences of known genes in other organisms
practical applications of DNA technology
diagnosis of diseases, gene therapy, pharmacuetical products, forensic science, environmental uses, agricultural uses,
restriction fragment length polymorphisms (RFLPs)
differences between nucleotide sequences among individuals (either in coding or non-coding regions of DNA; tend to differ more in junk than in coding)
cellulase
enzyme made by E.coli that allows cows and other animals to digest cellulose, making all plant parts usable as animal feed
yeast
eukaryotic, single-celled fungus with quick replication and plasmids
bacterial reproduction
exponential; occurs every 20 minutes
animal husbandry
farm animals treated with recombinant DNA products (vaccines, antibiotics, GH)
for what is PCR important?
for amplifying DNA for murder trials (blood, semen, tissue), amplifying DNA of very old cells (woolly mammoth), and determining genetic disorders from prenatal cells
concerns of DNA technology
genetic manipulations may create hazardous new pathogens; genes injected into organisms could be harmful to the organism or to organisms that come into contact with it (bacteria that clean up oil spills); new drugs may have harmful side effects; super weeds; how should info from the human genome project be used? gene therapy, but should a person's genome be a factor in job eligibility or for insurance purposes?; should we have the ability to genetically engineer our children?
environmental uses
genetically engineered bacteria can be used in mining minerals and cleaning up toxic mining wastes (bacteria can extract heavy metals from environment); microbes can degrade organic compounds into non-toxic form in sewage treatment plants (such as chlorinated hydrocarbons in water treatment plants, which are toxic and cannot be degraded naturally) and can be engineered to clean up toxic waste spills and dumps
problems associated with expressing cloned genes
getting a prokaryotic organism to express a eukaryotic gene is not a simple task; gene expression in both organisms is entirely different; problems with promoters and introns
bone marrow
gives rise to all blood cells and cells of immune system; prime candidate fir gene therapy, which can geneticalluy enhance these cells to improve the ability of the immune system to fight off cancer or genetically engineer immune cells to be resistant to HIV
super weeds
if genetically engineered plants cross-pollinate with wild neighbors, engineered plants may take over and not respond to weed control
the mapping of entire genomes
information obtained from studying individual genes allows researchers to pinpoint precise locations of all an organism's genes and non-coding segments along DNA molecules
making recombinant DNA
inserting genes into bacterial plasmids; restriction enzymes recognize restriction sites and cut the DNA at specific locations within the sequence; cut DNA produces sticky ends and DNA ligase seals the strands together with covalent bonds
2 useful purposes of recombinant DNA technology
it allows researchers to make multiple copies of genes for study or for transfer to other organisms (gene cloning); it allows proteins of interest to be produced in large quantities
what is the problem with in situ hybridization?
it is not absolutely specific; probes may recognize homologous DNA sequences, which may differ in a number of nucleotides
what is the value of yeast in overcoming eukaryotic/prokaryotic incompatibility?
it overcomes problems with promoters and introns
what is the problem with gene therapy?
it raises many ethical questions; should we treat human germ line cells (ovary, testicular cells) in hopes of correcting a defect in future generations?
genetic engineering
manipulation of genes for a practical purpose
biotechnology
manipulation of organisms for a practical purpose
why should scientists use eukaryotic cells instead of prokaryotic for gene expression?
many eukaryotic proteins will not function unless they are modified after translation by the addition of lipid or carb groups; bacterial cells cannot carry out these modifications, but eukaryotic cells can; many eukaryotic genes are too large to fit into bacterial plasmids
identification of the colony carrying the specific gene of interest
must happen once the bacterial colonies containing recombinant plasmids have been identified (white colonies)
nucleotide sequences and restriction enzymes
nucleotide sequences often repeat multiple times along a DNA molecule; therefore, one restriction enzyme my cut a DNA molecule numerous times
identification of desired gene
once bacterial cells have taken up recombinant plasmids, need to figure out which bacterial cells contain the plasmid with the desired gene, which can be identified by looking at the genes on the plasmid
transgenic organisms
organisms that contain genes from another species
example of gene cloning
pest resistance genes may be cloned and transferred to various plant species
genetic engineering in plants
plants have received genes for herbicide resistance (cotton) and pesticide resistance; genetically engineered fruits have been approved by the FDA, such as tomatoes engineered with antisense genes to retard spoilage
cloning vector
plasmid used when making recombinant DNA; molecule that carries foreign DNA into a cell and replicates there
expression vectors
plasmids that contain a highly active bacterial promoter upstream of the restriction site where the eukaryotic gene is inserted
sticky ends
produced by cut DNA; pair up with nucleotide sequences on the gene of interest to form recombinant DNA molecule
bacterial transformation
recombinant DNA technology; during transformation, a gene of interest is isolated and inserted into a bacterial plasmid, which is then inserted into a bacteria cell; as the bacterial cell replicates, the plasmid containing the gene of interest replicates
gene therapy
replacing non-functional or abnormal alleles with functioning ones
how do scientists overcome problems with introns?
scientists make artificial eukaryotic genes without any introns by converting mRNA (which already has introns taken out) into a single-stranded piece of DNA using reverse transcriptase; DNA polymerase then synthesizes a second strand of DNA which is complimentary to the first, making the DNA double-stranded
how do scientists overcome problems with promoters?
scientists use expression vectors, and the bacterial host will recognize the promoter and express the gene linked to it
forensic science
similarities or differences in small (1000 cell) samples of blood, tissue, or semen left at scene of a violent crime detected by RFLP analysis
antisense nucleic acid
single-stranded molecules of DNA or RNA that base pair with mRNA molecules and bloc ktheir transcription; could prevent spread of disease beyond the use of pharmaceutical drugs
restriction fragments
small pieces of DNA segments cut by restriction enzymes before gel electrophoresis
restriction sites
specific nucleotide sequences in DNA
DNA fingerprint
specific pattern of bands provided when radioactive probes that mark electrophoresis bands that contain RFLP markers, which usually test for 5 markers (only a few selected portions of DNA)
Sanger method of DNA sequencing
starts with many copies of a DNA fragment from with the nucleotide sequence can be determined; then uses sequencing machine
what is the speed/efficiency of DNA sequencing?
takes a very long time; by mid-1998 only 3% of genome had been sequenced
polymerase chain reaction (PCR)
the DNA to be copied, DNA polymerase, nucleotides, and short pieces of DNA to be used as primers (which start DNA replication) are mixed in a test tube; DNA polymerase can make billions of copies of a segment of DNA in a few hours
restriction enzymes
the enzymes that cut DNA molecules at specific sites and that are found in bacteria, which they protect from viruses by cutting up viral DNA
human genome project
the mapping of all of the genes in the human body from 1990-2003; also included mapping the genomes of other species: E.coli, saccharomyces cerevisiae (yeast), drosophila melanogaster (fruit fly), and mus musculus (mouse)
hybridization with radioactive probe
the probes hybridize with the restriction fragments and mark the specific genes or nucleotide sequences of interest
nucleic acid hybridization
the process used to find the colony with desired gene; involves making a single-stranded molecule of RNA or DNA with the nucleic acid probe
plasmid
tiny, circular piece of DNA that carries very few genes
what were the objectives of the human genome project?
to identify all of the 20,000-50,000 human genes and determine the base pair sequence of each of these genes
what can researchers do once multiple copies of a gene are made?
try to figure out the nucleotide sequence of the gene and how nucleotide sequences differ bwteen individuals
RFLP bands
unless identical twins, 2 individuals will not have the same of these
what is the role of fertilizers?
used to deliver nitrogen-containing compounds to plants that cannot undergo nitrogen fixation; if we can create bacteria that will fix nitrogen in corn or wheat or make plants capable of fixing nitrogen themselves, there would be no need for expensive fertilizers
yeast artificial chromosomes (YAC)
vectors created by scientists to combine an origin of replication, a centromere, telomeres, and foreign DNA which behave like ordinary eukaryotic chromosomes; useful because artificial chromosomes can carry much more DNA than plasmid
restriction fragment preparation
when DNA/RNA segments are mixed with restriction enzymes in a test tube, restriction enzymes cut the DNA into restriction fragments, which are then run through an agarose gel and sorted into distinct bands; this technique detects differences in the nucleotide sequences of 2 different individuals
photographic film
when the membrane is laid under this, the radioactive probes expose the film and create a black spot, which corresponds to the DNA that has hybridized to the probe and can be traced back to the original bacterial colony containing the gene of interest
special nylon membrane
where bacterial cells from each colony are transferred to get the nucleic acid probe to bond to the gene of interest; treated to break open cells and denature their DNA resulting in single-stranded DNA molecules, which stick to the membrane; it is then incubated in a solution of complimentary radioactive probe molecules which bond to the gene of interest
identifying which bacteria took up non-recombinant plasmids
will grow as blue colonies on agar containing ampicillin; can digest lactose because no foreign DNA has been inserted into the lac Z gene
identifying which bacteria took up recombinant plasmids
will grow as white colonies on agar containing ampicillin, since they cannot digest lactose because forein DNA has been inserted in the middle of the lac Z gene
identifying which bacteria have not taken up any plasmids
will not grow on agar containing ampicillin because they do not have amicillin resistance gene (ampR)