made possible: Human Genome Project completed in 9 yrs (2001)
by 2010 genomes of > 7,000 species
Slide 3
DNA formed when segments of DNA from 2 different species are
combined in vitro (in test tube) useful for analyzing genes &
gene expression
Slide 4
manipulation of organisms or their components to produce useful
products includes selective breeding, using bacteria in
fermentation
Slide 5
the direct manipulation of genes for practical purposes
Slide 6
useful for researcher to have just that portion of DNA working
with 1 gene may be as small as 1/100,000 th of a chromosome
bacterial plasmids: circular DNA molecules that replicate
separately from bacterial chromosome used by bacterium when
environment changes
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used to cut DNA w/in short, specific nucleotide sequences
(restriction sites) set of dbl stranded DNA fragments with single
stranded sticky ends
Slide 9
sticky ends form H-bonds with sticky ends of C bases from other
DNA: temporary bonds DNA ligase make bonds permanent: makes
covalent bonds in sugar- phosphate backbone
name given original plasmid dfn: a DNA molecule that can carry
foreign DNA into host cell & replicate there bacterial plasmids
mostly used because 1. readily available from suppliers 2. can
insert foreign DNA in vitro bacterial cell 3. multiply rapidly
large plasmids trimmed down so they contain just the genes
necessary for replication carry 100 300 kb (kilo base pairs),
normal plasmid can insert vectors no larger than 10 kb
Slide 17
can make libraries using cDNA:
Slide 18
each have advantages Genomic library good if: looking for a
gene but not sure where it is in a genome, or what kind of cell to
look in if looking for introns or regulatory sequences asc w/gene
cDNA library good if studying: specific protein sets of genes
expressed in particular cell types changes in patterns of genes
over life of cell (during development of organism)
Slide 19
Nucleic Acid hybridization: process of base pairing between a
gene & a C sequence on another nucleic acid molecule C molecule
= ssDNA or ssRNA = nucleic acid probe probe is made that is C to
known sequence in gene
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several technical difficulties hinder the expression of cloned
eukaryotic genes in bacterial hosts can substitute eukaryotic
hosts: yeasts, some insect cell, some mammalian cells that have
appropriate expression vectors: a cloning vector that contains a
highly active bacterial promoter just upstream of restriction site
where eukaryotic gene can be inserted allowing gene to be expressed
in bacterial cell or have been genetically engineered to use in
specific eukaryotic cells
Slide 23
Polymerase Chain Reaction amplifies specific target segment of
DNA in vitro using primers that bracket the derived sequence, &
a heat-resistant DNA polymerase, & nucleotides
once you have many copies of a gene you can ask questions about
its functions, where & when the gene is expressed, or how
important is it to the organism
Slide 28
uses a gel made of a polymer (agarose commonly) gel acts like
molecular sieve separates nucleic acids or proteins based on charge
and size nucleic acids carry (-) charges on phosphate group so (+)
end of gel as move the longer molecules impeded more by sieve
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fragments produced be restriction enzymes put thru gel
electrophoresis band pattern characteristic of starting molecule
& restriction enzyme used able to identify viruses &
plasmids by their band patterns then recover DNA from gels 1 way of
getting pure samples of DNA wont work with DNA from eukaryotic
cells: gel electrophoresis smear not bands
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Restriction Fragment Length Polymorphism : single nucleotide
polymorphism (SNP) that exists in the restriction site for a
particular enzyme, making the site unrecognizable by that enzyme
& changing lengths of the restriction fragments formed by
digestion with that enzyme found in coding & noncoding DNA
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used to detect certain nucleotide sequences w/in a complex DNA
sample compares the restriction fragments produced from different
samples of genome DNA
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method used to sequence relatively short DNA fragments done by
automated sequencing machines
Slide 38
technique: synthesizes a set of DNA strands C to original DNA
fragment each strand starts with same primer & ends with
dideoxyribonucleotide (ddNTP) incorporation of ddNTP terminates
growing DNA strand because it lacks 3 OH group (site of attachment
of next nucleotide) each ddNTP tagged with distinct fluorescent
label so identity of nucleotide at end of each strand (ultimately
entire strand) is identified
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in vitro hybridization with labeled probes looking for specific
mRNAs could be used to look at how expression of a gene changes
during the embryonic development of organism carry out gel
electrophoresis on mRNA
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Reverse Transcriptase- Polymerase Chain Reaction quicker &
more sensitive than Northern blotting isolates mRNA from different
developmental stages of organism then add reverse transcriptase to
make cDNA which serves as template for PCR amplification using
primers from gene being studied bands will be in samples that
originally contained the gene being studied
Slide 43
alternative method used to determine which cells are expressing
certain genes done in living organism probes labeled with
fluorescent dyes
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uncover gene interactions suggest correct therapeutic route in
cancer treatments
Slide 46
most common method used to determine function of gene: disable
it & observe what happens specific mutations introduced to
cloned gene & then mutated gene returned to cell knocking out
normal gene in the process
Slide 47
RNA interference method for silencing expression of selected
genes uses synthetic dsRNA molecules matching the sequence of gene
to trigger breakdown of the genes mRNA or to block its
translation
Slide 48
important when studying groups of genes to determine how
multiple genes interact (basis of systems biology, chap 21) in
humans considered unethical to block activity of genes
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used to analyze genomes of large #s of humans with certain
phenotype or disease test for genetic markers : DNA sequences that
vary in a population uses SNPs (single nucleotide polymorphisms)
single base pair site where variation is found in at least 1% of
population few million in human genome
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as advances being made in DNA technology also working on
technology to make multicellular organism from 1 cell producing
genetically identical organism 1 st attempted late 1950s
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Genomic Equivalence: an organisms cells have the same genome
proved when able to generate new organism from 1 cell used carrot
(root) cells cultured adult plant
Slide 56
totipotent: describing a cell that can give rise to all parts
of the embryo & adult, as well as extraembryonic membranes in
species that have them
Slide 57
used in cloning animals transplant nucleus from a
differentiated animal cell enucleated ova can sometimes give rise
to clone
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Embryonic Stem Cells (ES) or Adult Stem Cells from animal
embryos or adult tissues can reproduce & differentiate in vitro
and in situ ES cells are pluripotent : cell that can give rise to
many but not all parts of an organism difficult to acquire
Slide 60
ES cells currently donated by patients undergoing infertility
treatment or from long term cell cultures established with cells
isolated from donated embryos when main objective is to produce ES
to treat disease process called therapeutic cloning
Slide 61
now scientists can de-differentiate cells returning them to
pluripotent cells: called iPS: induces Pluripotent stem cells can
do anything ES cells can do
Slide 62
2 major uses 1. reprogram cells from patients with disease to
become iPS cells then act as model cells for studying the disease
& potential treatments Parkinsons disease, type 1 diabetes 2.
field of regenerative medicine patients own cells used to
regenerate damaged tissues
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introducing genes into afflicted individual (into somatic
cells) for therapeutic purposes useful for disorders caused by
single gene defect (overall, relatively small # of all diseases)
for it to be permanent, treated cells must be the ones that
continue to divide thru out patients life
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using Short Tandem Repeats (STRs) in DNA isolated from crime
scenes leads to genetic profile strong evidence to prove suspect
innocent or guilty used in paternity disputes identification of
remains
Slide 67
gene for desired protein inserted into bacterial genome and
become tiny factory for making protein Insulin Digestive enzymes
Growth Hormone
Slide 68
genetically engineered microorganisms developed for oil spills
or to degrade toxic waste materials Bacteria Algae Plants
