Download Chapter 12 Notes - Great Neck Public Schools

Chapter 12 – DNA Technology and the Human Genome
Bacteria as tools for manipulating DNA
I. Intro
A. Recombinant DNA technology (genetic engineering):
1. Combining genes from different species and putting
them into new cells (prokaryote or eukaryote) that will
express the genes.
B. Has and will have important applications
1. Mass production of useful chemicals
2. Creation of new strains of plants and animals
a) oil cleaning bacteria
b) pigs with transplantable organs
3. More efficient methods for doing basic and applied
research in the biological sciences
a) if you want to study a protein, put the gene in a
bacteria and have the bacteria make it.
II. In nature, bacteria can transfer DNA in three ways
A. In sexually reproducing organisms, new genetic
combinations are the result of meiosis and fertilization.
B. So how do bacteria produce new genetic combinations?
C. The DNA of most bacteria consists of a single
chromosome as a closed loop
D. Three ways that bacteria can take up DNA
1. Transformation – taking up DNA from the fluid
surrounding the cell
a) Frederick Griffith (1920’s) – showed that nonpneumonia causing strains of bacteria became
pneumonia causing when cultured in the presence of
dead disease-causing cells – scary!
2. Transduction – transfer of bacterial genes from one
bacterium to another by a phage.
3. Conjugation – bacterial cell mating
a) Males cells (gene donors) recognize female cells
(gene recipients) via the male sex pili.
b) After recognition, a cytoplasmic bridge spans the
two cells and replicated DNA from the male passes to
the female.
E. In all 3 cases, the new DNA is integrated into the
existing DNA by a crossover like event, replacing part of
the existing DNA
F. Important – these mechanisms are not reproductive,
sexual reproduction does not occur in bacterial
III.Bacterial plasmids can serve as carriers for gene
transfer
A. Conjugation relies on the presence of an F factor
B. F factor  “F” for fertility; a specific piece of DNA
1. Carries the genes for sex pili and other proteins
2. May exist integrated in bacteria chromosome or as a
plasmid
3. PLASMID  a small, circular DNA molecule separate
from the much larger bacterial chromosome
C. VECTOR  a plasmid that can carrying extra genes
other than those needed for replication and conjugation to
another cell
D. Bacteria can have many different kinds of plasmids
1. R PLASMIDS  “resistance” plasmids
a) Carry genes that destroy antibiotics like penicillin
and tetracycline.
b) Antibiotic Tx kills off bacteria lacking R plasmid
leaving resistant bacteria plenty of room to multiply
and antibiotics become useless.
IV. Plasmids are used to customize bacteria: An
overview
A. plasmids are first isolated from a bacterium
B. DNA carrying a gene of interest is obtained from another cell
C. A piece of DNA containing the gene is inserted into the plasmid
D. A bacterial cell takes up the plasmid by transformation
E. This genetically engineered, recombinant bacterium is then
cloned to generate many copies of the gene, which can be used
directly or translated into protein by the bacteria (human insulin)
V. Enzymes are used to “cut and paste” DNA
A. Restriction enzymes - bacterial enzymes that act as
scissors for making recombinant DNA in a test tube
1. RE’s are naturally found in bacteria as protection
against foreign DNA from other organisms and phages
2. RE’s cut at specific palindromic DNA sequences
a) give an example
B. The gene of interest and the plasmid are cut with the
same restriction enzymes allowing for the new gene to be
inserted into the plasmid.
C. DNA ligase – “pasting” enzyme – glue together the cut
ends – reforms the phosphodiester bond
D. The outcome is Recombinant DNA – DNA molecule with
a new set of genes
VI. Genes can be cloned in recombinant plasmids: A
closer look
A. Allows for the production of a desired gene on a large
scale
B. A biologist can create cells to produce desired proteins
in marketable quantities
VII. Cloned genes can be stored in genomic libraries
A. The first step of genetic engineering is isolation of the
gene of interest
B. Shotgun approach – cut up target DNA (human DNA for
example) into thousands of fragments using restrictions
enzymes.
C. Each fragments will contain one to a few unknown
genes – one or more fragments, however, will carry your
gene of interest
D. These fragments can be ligated into plasmids or phage
DNA and stored in bacterial cells or phage, respectively
VIII. Reverse transcriptase helps make genes for cloning
A. Problem with shotgun method above –
1. eukaryotic genes contain introns! Need to get rid of
introns if you want a bacteria to make your protein
(bacteria don’t know what introns are).
2. The fragments might be too long with introns – hard to
work with long fragments
3. Many of the fragments will be meaningless – no genes
B. Solution – Remember HIV? – reverse transcriptase
1. Instead of purifying the cells DNA, we will grab the
mRNA – no introns!
2. RT can transcribe mRNA back to DNA and the ssDNA
can be made double stranded using DNA polymerase! –
this DNA is called complementary DNA (cDNA) because it
complements the RNA.
3. These “clean” (intron-less) fragments can then be
stored just as before in bacteria of phage.
C. Another advantage – your only getting the genes active
in the specific cell type you are interested in. If you use
nerve cells, you will only get genes that are active in nerve
cells, etc…
IX. Nucleic acid probes identify clones carrying
specific genes
A. Now how to fish out the gene you want from your new
library?
B. Probe – a radioactively labeled single-stranded DNA that
will be used to find a specific nucleotide sequence within a
mass of DNA
C. The probe in complementary to the sequence of interest
as to pair with the desired gene.
X. DNA microarrays test for the expression of many genes at
once
A. DNA microarrays – allows scientists to determine which
genes are being transcribed in particular cells at particular
times.
1. Isolate mRNA from cells
2. Make fluorescently labeled cDNA of mRNA
3. Test cDNA mixture for base pairing with DNA from
different genes.
XI.
Gel electrophoresis sorts DNA molecules by size
A. Gel electrophoresis - a method for physically sorting
macromolecules – proteins or nucleic acids – primarily on
the basis of their electrical charge and size
B. The gel has tiny pores that the DNA must travel through
to get to the other side
1. Small pieces get through holes easier and hence go
faster
C. Use electricity to move the DNA fragments – DNA is
negatively charged due to its phosphates so they move
towards the positive pole.
XII. Restriction fragment analysis is a powerful method
that detects differences in DNA sequences
A. Genetic marker – any (doesn’t have to be a gene) piece
of DNA the VARIES from person to person
B. The DNA amongst related individuals is more likely to
match than between unrelated individuals
C. Restriction fragments – the pieces of DNA that result
from cutting DNA up with restriction enzymes
1. Different people will have different restriction
fragments
2. Where restriction enzymes cut depends on your DNA
sequence. Thus, the fragments of relatives will be more
similar than non-relatives
D. Detecting harmful alleles in heterozygotes
1. Diseased allele usually carries on or more restriction
sites not found in the normal allele – more restriction
fragments
E. Restriction fragment analysis only needs 1mg of DNA (a
drop of blood). But what if you don’t have that much DNA?
XIII. The PCR method is used to amplify DNA sequences
A. PCR = polymerase chain reaction – a technique for
amplifying any segment of DNA in a test tube without the
use of living cells.
B. A mixture of DNA, DNA polymerase, and nucleotide
monomers will continue to replicate, increasing the amount
of the desired DNA segment exponentially.
C. Has revolutionized DNA work
1. Was used to amplify DNA from an ancient mummified
human, a 40,000 year old wooly mammoth, a 30 million
year old plant fossil
THE CHALLENGE OF THE HUMAN GENOME
XIV. Most of the human genome does not consist of
genes
A. 97 % of the 3 billion bases is non-coding
B. Most of which is “JUNK” DNA
C. “JUNK” DNA really means we don’t know its function
D. REPETITIVE DNA  nucleotide sequences present in
many copies in the genome
E. TELOMERES  repetitive DNA at the chromosome
ends; may have a protective function
F. JUMPING GENES = TRANSPOSONS – discovered by
Barbara McClintock in the 1940’s
1. Segments of DNA that can move from one location to
another with a chromosome or between chromosomes
2. May aid in genetic diversity and evolution
XV. The Human Genome Project is unlocking the
secrets of our genes
A. An effort to map the entire human genome (completed in
2005)
B. In 2002, the genomes of over 70 organisms had been
sequenced
C. Remember, DNA is just a parts list. Someone needs to
figure out what all of these parts do!
OTHER APPLICATIONS OF DNA TECHNOLOGY
XVI. DNA technology is used in courts of law
A. Everyone with the exception of identical twins has a
different DNA sequence
B. DNA fingerprinting –
1. Restriction fragment analysis on about five genetic
markers
a) Only need about 1000 cells (tiny amount) taken
from blood or other tissue at the scene of a crime.
2. PCR repetitive DNA loci
XVII. Recombinant cells and organisms can massproduce gene products
A. Bacteria are the host of choice for making large
amounts of gene products
1. Simple
2. Grown rapidly and cheaply
B. Saccharomyces cerevisiae – single celled fungus and is
the cell of choice if one needs to use a eukaryotic cell to
produce gene products (rapid and cheap) - there are yeast
plasmids.
C. Some gene products are best made by mammalian cells
(insect cells lines are usually the starting point).
XVIII.
DNA technology is changing the pharmaceutical industry and medicine
A. Therapeutic Hormones
1. Human Insulin (Humulin) – one of the 1st commercially
produced recombinant DNA products – more effective
than insulin from pigs and cattle.
B. Diagnosis and treatment of disease
1. PCR to identify HIV
2. Identification of harmful alleles
C. Vaccines – harmless or derivative variations of proteins
produced on the surface of pathogens.
1. Mass produce vaccine proteins (Hepatitis B vaccine)
2. Make a harmless mutant of the pathogen
3. Replace proteins on surface of the harmless smallpoxlike virus that was used to vaccinate against and
eradicate smallpox in the 1970’s with proteins from a
number of other pathogens.
XIX. Genetically modified organisms are transforming
agriculture
A. Nitrogen fixation – conversion of atmospheric nitrogen
(N2) to biologically useable nitrogen, ultimately amino
acids.
1. Nitrogen fixing genes are found in a few bacteria living
in soil, roots, lichens, and aquatic habitats.
2. What if we could take these genes and put them into
plants? Not as simple as it sounds because plants lack
the specific environment of the bacterial cells where
these enzymes normally function.
B. Delayed ripening and disease resistance has been
improved in plants.
C. So how do we get genes into plants? – Fig. 12.18A
1. Agrobacterium tumefaciens –
a) a pathogen to a number of plant hosts.
b) Used to transfer genes to plants in recombined
form with the bacterium’s Ti plasmid resulting in a
TRANSGENIC ORGANISM
c) Problem is that A. tumefaciens does not grow in all
plants like grains.
D. GENETICALLY MODIFIED (GM) ORGANISMS  acquired
one or more genes artificially rather than breeding
E. Transgenic animals – do exist, very useful in research:
1. Transgenic mice have been made that are susceptible
to HIV, which will help with AIDS related research.
XX. Gene therapy may someday help treat a variety of diseases
A. Gene therapy – alteration of an individuals genes
B. Many ethical questions:
1. Who will have access? Expensive
2. Should it be reserved for only the most serious
conditions?
3. What about for athletic ability, physical appearance, or
intelligence?
C. Its even easier to modify genes zygotes
1. Should we eliminate genetic diseases?
2. Now we are interfering with evolution… or are we?
3. Eliminating unwanted alleles could backfire – reduces
genetic variety, damaging genes under one condition may
be necessary under another (sickle cell anemia)
RISKS AND ETHICAL QUESTIONS
XXI. Could GM organisms harm human health or the
environment?
A. Genetic engineering involves risks
B. It could produce new pathogens
C. What if a human oncogene found its way into a
lysogenic virus?
1. Safety guidelines have been established and
administered by the US government –
a) Often require these organisms to be genetically
altered such that they would not be able to survive
outside the lab.
b) Forbidden to work with human cancer genes or
genes of extremely virulent pathogens.
2. Controversy have erupted nevertheless
a) Frostban
D. Most of the public concern today has been on
genetically modified (GM) crops
1. Are crops carrying genes from other organisms
hazardous to out health?
E. Example – Bt corn and the Monarch butterfly
XXII. DNA technology raises important ethical questions
A. Should we be creating new organisms and adding them
to the environment?
B. Should we be modifying our own species?
C. What will be the implications to the ecosystem?
D. For what will we use this technology…medicine or war?
E. Who will benefit and in what way?
F. Eugenics – the effort to control the genetic makeup of
human populations
1. Our society rejects the notion of eugenics largely
because of Nazi Germany
2. Our hands are not clean, however, eugenics was
practiced in the US…recently!
G. Genetic discrimination