DNA Technology and Development Flashcards
Recombinant DNA
DNA from 2 different sources that is combined
Genetic Engineering
direct manipulation of genes for practical purposes
In vitro
in glass
in a test tube or flask
biotechnology
manipulation of organisms to perform practical tasks or provide useful products
gene cloning
making well-defined gene sized pieces of DNA in multiple identical copies
restriction enzymes
enzymes that occur naturally in bacteria that protect the bacteria against intruding DNA from other organisms
how do restriction enzymes work?
work by cutting foreign DNA at specific points along the DNA strand
what does a specific restriction enzyme recognize?
a restriction site
what is a restriction site?
specific short nucleotide sequences
what does it mean when most restriction sites are palindrome?
they have the same order of nucleotides read either forward or backward
how long are restriction sites?
4-8 nucleotides
what does the restriction enzyme cut through?
covalent phosphodiester bonds and H bonds of both strands
sticky ends
the fragments of DNA with single stranded DNA made from cuts
since one type of restriction enzyme always cuts at the same restriction site, how are the restriction fragments?
they are always the same
can a restriction enzyme make many cuts?
as long as the target sequence is there, the enzyme will cut all of them
how to use bacteria to clone genes?
Isolation of plasmid DNA and DNA containing gene of interest
gene inserted into plasmid =recombinant DNA (vector)
plasmid put into a diff bacterial cell
cells clones with gene of interest
identification of desired clone
various applications - copies of gene or protein
What happens when the sticky ends are made?
form H bonds with their complementary base pairs, bringing about recombinant DNA molecule if it is from another source
what seals the strands together?
DNA ligase, forming covalent phosphodiester bonds
what are the problems that arise with getting a prokaryote to function with eukaryotic DNA in it?
prokaryotes lack introns, while eukaryotes have them
how have scientists avoided the intron problem with prokaryotes and eukaryotes?
make artificial eukaryotic genes that lack introns by allowing a cell to undergo transcription and make mRNA, then allowing splicesome to remove introns
what happens after RNA splicing?
mRNA is added to a solution of reverse transcriptase which creates a strand of DNA minus the introns
what is the “intron free” DNA called?
complementary DNA (cDNA) that can now be inserted into a vector
what do scientists also use to avoid prokaryotes-eukaryotes incompatibility?
yeast as they are single-celled fungi that grow as easily as bacteria and have plasmids and are eukaryotes
what are the artificial chromosomes that scientists have made?
that are vectors containing an origin of replication, a centromere, 2 telomeres, and most importantly foreign DNA
how at the artificial chromosome vectors compared to plasmid vectors?
much longer enabling long pieces of DNA to be cloned`
why is it better to use eukaryotic host cells for expressing a cloned genes?
many proteins are altered after translation by the addition of a lipo- or glyco- proteins, using host cells from an animal or plant culture may be necessary
how can eukaryotic cells use electrical impulses to allow DNA to enter?
electroporation - use of brief electrical impulses that create temporary holes that allow DNA to enter
how else can scientists inject DNA into an eukaryotic cell?
microscopic needles
PCR
Polymerase Chain Reaction - used to clone scanty or impure DNA
how many clones can PCR make?
billions in a few hours
what does PCR start with?
special kind of DNA polymerase (Taq), a supply of DNA nucleotides, single stranded DNA primers, and the DNA to be cloned in a test tube
how is the DNA prepared in PCR?
heated to separate the strands, then DNA primers hydrogen bond each strand, and DNA polymerase adds nucleotide in its 5 to 3 direction.
what clones the DNA in PCR?
the heating again and cooling cycle that repeats over and over
steps of PCR
Denature
Anneal
Extend
how are the DNA clones of PCR?
in small amounts/partially degraded
DNA fingerprints
DNA fragments
what is electrophoresis used for?
to analyze DNA by separating DNA/proteins into bands on the basis of sizes and electrical charge
what is Restriction Fragment Analysis used for?
compare different alleles of a gene or DNA of different individuals or species
what does Restriction Fragment Analysis involve?
involves treating the DNA molecules in question with restriction enzyme then doing gelelectrophoresis which yields different banding patterns RFLP
what is RFLP?
restriction fragment length polymorphism - when homologous chromosomes vary in length of fragments due to different cuts by restriction enzymes
why do the cuts of restriction enzymes vary?
homologous chromosomes can be different alleles
what happens after gel electrophoresis in restriction fragment analysis?
fragments are further analyzed by southern blotting
what is southern blotting?
putting gel from restriction fragment analysis in an alkaline solution and putting blotting paper and denatures it by breaking H bonds
then paper is exposed to a single stranded radioactive nucleic acid probe that forms complementary base pairs to the DNA on the blotting paper
how is restriction fragment analysis complete?
by exposing the radioactive probes to photographic film which yields specific DNA bands
Human Genome Project goal?
find the precise location of all an organism’s gene and introns
what did researchers map in the Human Gene Project?
genomes of humans, Ecoli, yeast, Drosophila, and mice
what can the mapped genomes confirm?
DNA sequences can be compared and confirm evolutionary connections between even distantly related organisms
3 domains of life/superkindgoms
Archaea, Bacteria, Eukarya
what did the completed genomes of the 3 domains of life support?
the theory that they represent the superkingdoms of life
what is biotechnology making enormous contributions to?
diagnosing diseases
how is biotech helping with diseases?
hundreds of human genetic disorders have been identified before the onset of symptoms
carrier of potentially harmful alleles can be identified as well
what does Human gene theory allow for?
enables scientists to correct genetic disorders by replacing a defective allele with a functional one using recombinant DNA techniques
what needs to happen for gene therapy of somatic cells to be permanent?
the cells tat receive the normal allele must ne ones that multiply throughout the patient’s life
where can the new allele in gene therapy be inserted?
into the somatic cells of the tissue affected by the disorder in a child/adult or even the germ line cells (make gametes) or embryonic cells
downside of gene therapy
ethicality and may lead to eugenics
eugenics
an effort to control the genetic makeup of human populations
what type of pharmaceutical products has DNA technology created?
proteins
pharmaceutical products has DNA technology created for diabetics? correct dwarfism? reduce risk of heart attacks?
insulin
human growth hormone
tissue plasminogen activator
what is the downside with the genetically engineered products?
developmental costs are high
market isnt broad
products are expensive
why is DNA technology used in crime scenes?
every individual’s DNA is unique (except identical twins)
what is RFCP used for in crime scenes?
common method to detect similarities and differences in DNA samples and only requires tiny amounts of blood or tissue
DNA fingerprint
small portions in DNA that are highly variable from person to person
Transgenic organisms
organisms that contain genes from another species, have also been developed by injecting foreign DNA into nuclei of egg cells or early embryos
how have farming animals been treated with products made by recombinant DNA methods?
dairy cows injected with rBGH (recombinant bovine growth hormone) made by E.coli to raise milk production
BGH also improves weight gain cattle
GMO
genetically modified organism
how are plants easier to genetically engineer?
with most plants an adult can be grown from one cell
what is the vector commonly used to move genes into plants?
plasmid from the bacteria Agrobacterium tumefaciens, and not E.coli
what does the bacterium do?
infects and causes tumors called Crown Gall Disease
crown gall causing plasmid
Ti (tumor inducing) plasmid
what must scientists do when they insert a new gene into a plant using the Ti plasmid?
they must remove the genes that cause the disease and replace it with foreign DNA
what do many of the genetically engineered plants have genes for?
herbicide resistance
how does herbicide resistance help farmers?
helps to control weeds and not destroy crops
what are many crop plants being engineered to resist?
insects, enabling farmers to reduce the need of chemical insecticides
what are many fruits being genetically engineered to do?
contain genes that retard spoilage
how is the slowing of spoilage done?
done by blocking the production of the ripening hormones (ethylene) so plants only produce less of these
what does a single celled zygote undergo to give rise to many cells?
mitosis/cleavage
are the cells that are risen the same?
no, they have differentiated into specialized kind of cells with specific structures and functions
how are cells organized? system of organization?
cells- tissues - organs - organ system - organisms
morphogenesis
the process that gives rise to the shape of the organisms and its parts
what does morphogenesis lay out?
the body plan of an organism very early in the embryonic development
problems in morphogenesis can lead to?
cleft palates
most research done on development in done using model organisms such as?
drosophila
nematode C elegans
mouse
zebrafish
plant Arabidopsis
what is the criteria for a model organism?
readily observable embryos
short generation times
small genomes
preexisting knowledge about the organism’s genes
how can a whole new plant be grown?
from differentiated somatic cells since they are totipotent stem cells
totipotent
cells retain the ability to form all parts of the adult organism
do differntiated animals cells develop into a new organisms?
no
how must you clone an animal cell?
the nucleus of an unfertilized egg is removed and then fused with a somatic cell from the animal to be cloned. normal mitotic divisions produce an embryo and then the embryo is inserted into a surrogate mother’s uterus where after gestation, an animal identical to the donor is born
first cloned animal?
Dolly - a sheep containing chromosomal DNA identical to that of the nucleus of the donor in 1997
what is the first evidence of differentiation?
differentiated cells make tissue-specific proteins
what do muscle cells make?
myosin and actin
what do muscle cells fuse to form?
an elongated, multinucleated skeletal muscle fiber that goes into the Go phase and stops the cell cycle
where is the first difference that arises amongst cells in an embryo?
is from the cytoplasm of the unfertilized egg cell
the cytoplasm contains proteins and organelles from who?
encoded by the maternal DNA
cytoplasmic determinant
with each mitotic division, the cytoplasmic environment is different for each cell and this regulates the expression of genes affecting the fate of cells
induction/inductive signaling
chemical signals between cells cause changes in nearby cells which in turn induces target cells to differentiate into specialized cells
pattern formation
the spatial organization of tissues and organs develop in their characteristic places by this
when does pattern formation begin?
early in the embryo when the animal’s basic body plan of 3D structure is arranged
construction of body plan of Drosophila
head, thorax, abdomen
the cytoplasmic determinants in the unfertilized egg do what?
provide information about the 4 axes
4 axes
dorsal, ventral, anterior, posterior
egg polarity genes
mother has these that code for cytoplasmic proteins to ensure the embryo’s axes line up
what polarity protein do Drosophila have? what does it do?
Bicoid- anchors on the anterior end of the embryo and tell the developing organism “this end up”
what had the study mutants in Drosophila led to?
a broad understanding of development
defects such as extra set of wings or legs on fruit fly’s heads had helped researchers with what?
develop a genetic map or locations of specific genes
embryonic lethal mutations
mutations leading to death at the embryo or larval stage
how are fruit fly structures developed on the appropriate segments?
regulatory genes called homeotic genes
what do the homeotic genes code for?
code for specific transcription factors and specify the types of appendages that will form and their
what do the homeotic genes code for?
code for specific transcription factors
three things homeotic genes do?
specify the types of appendages that will form
their correct location
and body segments
what is interesting about the homeotic genes?
these genes are very similar throughout all kingdoms
how long are homeotic genes in Drosophila?
180 nucleotides
what are the homeotic genes called?
Homeobox
what does the similarity of the homeobox gene sequence show?
it evolved very early in life and has been conserved virtually unchanged for millions of years
do all homeobox control the identity of body parts?
no, but most are involved with development
apoptosis
programmed cell death
caspases
suicide proteins
what happens in the cells destined to die in development?
cells trigger the activation of caspases
what do caspases do next?
trigger the release of enzymes like proteases and nuceleases
what happens to the targeted cells?
they shrink
nuclei condenses
get engulfed and digested by neighboring cells - phagocytosis
apoptosis is essential for normal development of what?
the nervous system
lymphatic system
morphogenesis of human hands and feet preventing ebbed fingers and toes
how is apoptosis linked to cancer?
in a normal cell with damaged DNA should trigger apoptosis, but cancerous cells continue to survive
