Chemical Basis of Molecular Genetics

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Chemical Basis of Molecular Genetics
molecular biology uses only a few key concepts
for all of the work fit ideas together, and
technique are easier to understand A.
complimentary sequences bind to each other
2 strands of nucleic acids WANT to stick together
(ie. sticky ends) B. enzymes that work in cells
work in test tubes too DNA polymerase
synthesizes DNA from primers DNA ligase
ties 2 pieces of DNA together restriction
enzymes cut DNA at particular bases
recombinases/transposases work on natural and
manipulated DNA C. labeling one thing allows
anything bound to it to be identified D.
separation by size using electrophoresis E.
transfection of DNA into other cells
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Restriction Endonucleases
Restriction endonucleases enzymes which cleave
within sequences of DNA at particular
sequences usually cleave palindromic
sequences palindrome sequence that reads the
same way in both directions ie. complementary
sequence is identical to the other side
GGGCCC restriction site for Sma1
(blunt) CCCGGG restriction site for Xma1
(sticky) most restriction enzymes are protein
homodimers, forcing them to cut palindromic
sequences restriction fragment piece of DNA cut
by restriction enzymes at both ends
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Restriction Endonucleases
sticky end-- cleaved piece of DNA with a short
overhang of single stranded DNA (that can be
used for sequence specific DNA ligations) G
GGCCC note the single stranded DNA is
complimentary CCCGG G blunt end-- cleaved
pieces of DNA without overhanging nucleotides
(any 2 cut pieces can be joined together using T4
or 7 DNA ligase) GGG CCC CCC GGG
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Restriction Endonucleases
restriction enzymes generate fragments of
different sizes from DNA mutations or
polymorphisms within the DNA sequence can change
the size of a fragment fragments can be cut
out of an agarose gel to purify them from
other pieces there are many different
restriction enzymes, many with different
sequences of DNA that are cut (most commonly 6
base palindromes) restriction map pattern of
restriction sites in a given DNA sequence
6kb
5kb
4kb
3kb
2kb
size polymorphisms
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DNA hybridization
agarose gel electrophoresis separates DNA pieces
by their size small pieces migrate faster than
large ones
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DNA hybridization
denaturation separation of 2 strands of
DNA renaturation coming together of 2 strands
of DNA (not necessarily the same 2 strands
that were denatured!) DNA needs to be
transferred to a nylon filter (positively
charged) from an agarose gel, usually done by
blotting (either wicking action or
electrophoretically) probe piece of nucleic
acid labelled so it can be detected
later hybridization incubating a probe with a
filter bound with DNA to identify a piece of
complimentary DNA
all DNA
labeled DNA
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DNA hybridization
1a) Separate nucleic acids 2a) transfer to a
nylon filter 3a) denature DNA on filter 4a)
block filter so probe can't bind
1b) obtain a nucleic acid probe 2b) label
nucleic acid (32P, biotin, digoxigenin,
fluorescein, etc) 3b) make sure probe is
denatured
5ab) mix labeled probe nonspecific DNA with
denatured DNA on filter 6ab) wash excess probe
away using (often) temperature to control
binding low stringency allow probe with
related sequences to stay bound high
stringency only allow very close matched
sequences to stay bound 7ab) detect labeled
probe-- identifies specific sized pieces of DNA
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DNA hybridization
Southern blot pieces of DNA separated on a gel
after restriction digest and probing using
some labeled nucleic acid (usually DNA) Northern
blot RNAs are separated on a gel and hybridized
with a probe
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DNA hybridization
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Cloning Making Many Copies of One DNA
cloning taking a piece of DNA and ligating it
into a self-replicating piece of DNA (usually
a plasmid or bacteriophage) plasmid independent
DNA circle that replicates separately from rest
usually present in multiple copies per cell
different plasmids can replicate in bacteria or
eukaryotes or both modern plasmids contain
many restriction sites that can be used to
clone in many different sequences for various
purposes different plasmids are for DNA
sequencing, protein overexpression,
transgenic organisms, eukaryotic protein
expression even with restriction digesting and
hybridization, it is difficult to clone the
specific genomic DNA that you're interested in
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Reverse Transcriptase
RNA viruses use RNA as their primary genetic
material-- ie. HIV these viruses have a very
special enzyme called reverse transcriptase
which can make a DNA copy from the RNA Molecular
biologists use reverse transcriptase to make
'copy' DNA or cDNA (DNA made from
mRNA) allows scientists to study exactly what
mRNA (i.e. proteins) are made
reverse transcriptase binds DNA, makes 1
strand many times over
primer DNA polymerase makes double stranded DNA
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cDNA
cDNA is a tool that is commonly used by molecular
biologists ie. isolate messenger RNA using its
polyA tail-- specific for mRNA make cDNA from
that mRNA using reverse transcriptase ligate
cDNA into a vector (usually a bacterial plasmid
or bacteriophage) put that new recombinant
DNA into bacteria by transfection now you
have foreign, maybe human, DNA in the bacteria
for screening by hybridization library
collection of foreign DNA cloned into a vector
for isolation and analysis many different
sizes and sequences
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Polymerase Chain Reaction PCR
relies on several key features to work 1)
small pieces of DNA can hybridize at moderately
high temperatures 2) must have the DNA sequence
to design synthetic primers 3) use a DNA
polymerase that withstand high temperatures
which are able to denature the DNA
strands first identified from Thermus
aquaticus, a bacteria found in thermal pools
now at least half a dozen thermostable
polymerases for sale PCR amplification is
exponential-- newly synthesized strand can be
used as a template for the next cycle of
reactions oligonucleotides (short DNA pieces)
can be chemically synthesized
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Polymerase Chain Reaction PCR
many primers and polymerases around
separate and hybridize
extend from the primer
separate and hybridize
2nd round, 4 oligos ready to go
extend from the primer
all new strands will be templates next cycle-
exponential
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How to clone your DNA
figure out what DNA you are interested in
genome sequences, related species sequences,
messenger RNA from a particular tissue obtain
DNA for what you want (reverse transcribed RNA,
PCR primers, etc.) make PCR primers with
desired restriction sites at both ends and
amplify desired DNA coding for what you want
(usually a protein)
note the new longer size!
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How to clone your DNA
restriction enzyme sites at the ends of the PCR
primers make cloning much easier-- allows
fast, accurate movement to another piece of
DNA find a restriction seqeunce that is NOT in
the piece of DNA you are cloning-- otherwise
you will cut your PCR band in the middle as
well choose your restriction site based on
what vector you are cloning into vector another
DNA molecule which can accept a piece of DNA a
carrier for your piece with specific
properties sticky ends (asymmetric cutting) are
better than blunt-- ligation is more
efficient, uses short overlap for specificity
EcoR1
EcoR1
cut with EcoR1 and run an agarose gel
uncut cut
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How to clone your DNA
plasmid particular type of circular DNA vector
most commonly used in DNA cloning experiments
usually 3-6 kb long will usually have at least 1
multi-cloning site region of the plasmid that
has been engineered to include a lot of different
unique restriction sites this gives lots of
opportunities to clone your DNA into the vector
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Plasmid maps
plasmids have several requirements that allow
them to be used
bacterial origin of replication
eukaryotic promoter
antibiotic resistance gene (kanamycin/ neomycin)
multicloning site
polyadenylation sequence
prokaryotic/ eukaryotic promoter
eukaryotic origin of replication
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How to clone your DNA
cut plasmid and insert (what you are cloning)
with 1 or 2 restriction enzymes gel purify your
cut DNA on an agarose gel ie. run a gel, then
cut your band out of it mix your cut vector with
insert and DNA ligase DNA ligase enzyme which
joins 2 fragments of DNA-- sticky ends are more
efficient T4 DNA ligase viral enzyme that can
stick blunt ends of DNA together
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Recombinant DNA
with DNA ligase, it's possible to join any 2
pieces of DNA together for example, if you want
to make an entirely new protein that is a
hybrid or chimera of two other proteins, you
design your two PCR pieces, ligate them
together into a vector and off you go ie there
are a group of proteins isolated from jellyfish
that fluoresce it's possible to fuse a protein
of interest to a naturally fluorescent
protein and watch what happens to that protein in
a living cell ie. can you change where the
protien is localized
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Transfection
transfection process of putting foreign DNA into
another cell three common ways of doing this
1) electroporation using an electric field
to shock cells into taking up the
foreign DNA-- extremely efficient 2) heat
shock raising the temperature of
specially treated bacteria so that they
take up the foreign DNA 3) lipofection
using a mixture of lipids bound to DNA
that will fuse with the cell membrane,
releasing DNA inside cell
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More DNA Vectors
cosmids are similar to plasmids with circular DNA
but have some bacteriophage sequences which
allow them to go up to about 50
kb bacteriophage virus that infects bacteria l
phage are the most commonly used-- usually for
making libraries very useful because they can
be purified to high titre (particles/mL) lent
ivirus RNA virus similar to AIDS manipulated
to be unable to reproduce AND to infect almost
any cell commonly tested for in vivo
uses artificial chromosomes several varieties,
including yeast and bacterial basically very
large recombinant DNAs-- 300 kb not uncommon
in yeast, have centromeres, rep. origins, etc
like any other chromosome
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8 Step Program for Cloning DNA
1) Design PCR primers (with restriction sites)
that amplify your DNA use restriction sites
that will not cut in your plasmid or DNA! 3) cut
the plasmid and the PCR fragment with restriction
enzymes 4) separate the cut pieces on an agarose
gel-- cut out appropriate bands 5) mix pieces
and ligate together using DNA ligase 6)
transfect the ligation reaction into bacteria
select for transfected cells using antibiotic
resistance 7) grow up the bacteria and purify
your new plasmid 8) make sure the plasmid is
correct by DNA sequencing
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dideoxy DNA Sequencing
polymerases use a free 3' hydroxyl to add a new
dNTP to a growing DNA chain, releasing 2
phosphates dideoxynucleotides end DNA synthesis
no 3' hydroxyl for the next dNTP to
bind often run 4 reactions, one each with
ddA, ddC, ddG, and ddT to stop randomly at
each base label each ddNTP with 35S or
fluorescence
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dideoxy DNA Sequencing
combining dideoxy nucleotides with PCR, can
sequence small amounts of DNA-- linear
amplification of pieces instead of
exponential newer method- use different
fluorescent colors representing the different
base can have all 4 ddNTPs in one lane- gives
more sequences per gel, with 1 sequence per
lane
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dideoxy DNA Nucleotides
Bacterial and viral polymerases are somewhat
different than eukaryotes some viruses use RNA
as their genetic material-- note that this
requires an enzyme that can recognize RNA as a
genetic template reverse transcriptase enzyme
that synthesizes DNA from RNA template
because it is a polymerase, it is required in
the viral life cycle
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dideoxy DNA Nucleotides
some dideoxynucleotides have fairly high affinity
for the polymerase ie. AZT, DDI are nucleotide
analogs that block RNA polymerases
(AIDS) analog molecule that is related to the
real one, but can't undergo a particular
reaction ie. transition state analogs dideoxynuc
leotides are common drugs to fight AIDS- by
blocking reverse transcriptases
preferentially, viruses can't replicate and cells
can
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Genomic Sequencing
Fragment the human genome using X rays-- clone
large fragments into artificial
chromosomes order the artificial chromosomes
and subclone into cosmids order cosmids along
the artificial chromosomes sequence DNA from
cosmids in a random fashion-- shotgun
sequencing put lots of overlapping fragments
into a computer to sequence a cosmid use
overlapping cosmid sequences to map artificial
chromosomes use overlapping artificial
chromosomes to generate a complete sequence
build up overlapping short sequences
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Reverse Genetics
Mendel started with his mutant phenotypes to come
up with his rules Today, we know DNA sequences
of many genes but not their phenotype reverse
genetics generation of an organism expressing
your desired gene easiest way-- engineer a
retrovirus to express your gene via
cloning infect embryonic stem cells with your
retrovirus-- integrates randomly into genome
as part of the retroviral life cycle, then inject
into embryos
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Reverse Genetics- Transgenics
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Reverse Genetics
gene targeting procedure for introducing a
specific mutation into 1 gene in the
organism first, obtain the genomic DNA sequence
of your gene of interest next engineer a large
plasmid with your desired mutation in that
genomic DNA (stop codons, deletions,
insertions, entire replacements, etc)
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Reverse Genetics
transfect embryonic stem cells with your plasmid
and look for ES cells that have undergone
recombination (usually using PCR or
hybridization looking for restriction fragment
length polymorphisms) using antibiotic
resistance inject your mutant ES cells into
blastocysts as with retroviruses
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Reverse Genetics
rescue ability of a cloned fragment of DNA to
recover a wild type phenotype in the
organism ie. putting back a deleted gene to
recover full function transformation rescue use
of genomic DNA sequences to reproduce the
expression pattern of the wild type
gene regulatory promoter and enhancer regions
are difficult to identify-- generally
relatively small and spread out compared to
bacteria minimal regulatory region the
smallest amount of DNA to reproduce the normal
pattern
mRNA
regulatory region (enhancers and silencers)
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Applications of Genetic Engineering
growth hormone is a small protein that can
increase the size of an animal you can make a
transgenic animal overexpressing growth
hormone can also inject human growth hormone
made in bacteria into humans as a treatment
for dwarfism insulin for treating diabetes is
another protein that can be made in bacteria
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Applications of Genetic Engineering
plants can also be engineered -- GM (genetically
modified) crops other strains of plants have
been generated to be resistant to a particular
herbicide-- now you can spray a field with that
herbicide and not kill your crops while killing
weeds other plants express a natural
pesticide-- kills insects that try to eat the
plants What do these new organisms mean for
human consumption/health?
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Applications of Genetic Engineering
rice has been designed to make b carotene using 4
genes from 2 different organisms-- daffodils
and bacteria 400 million people are deficient
in b carotene (Vitamin A) 'ice-minus' bacteria--
live symbiotically with the plants and provide
an 'anti-freeze' protein to protect the
plants from frost
drought resistant rice survives much better--
may increase yields 20 rice is a major choice
for engineering because so many people eat it
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Applications of Genetic Engineering
gene therapy use of recombinant DNA (often
viruses) to correct genetic defects has been
used successfully to treat severe combined
immunodeficiency syndrome (SCID)
works best in stem cells that will give rise
to well understood progeny harder with things
like neurons-- must become part of an
existing circuit being tested with Parkinsons,
Alzheimer's, stroke patients
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Functional Genomics
functional genomics expression pattern of all
the genes in all the tissues of the body and
all the normal and disease states ie. changes
in gene expression during development, aging,
cancer, etc only possible with the sequencing of
the human genome 25,000 genes northern blotting
was the hybridization technique of separating all
the mRNA on a gel, blotting it, and using a
probe to see a single mRNA microarrays reverse
the blotting process-- take all 25,000 genes and
put them in 25,000 different spots (each very
small) according to a pattern (do it in at
least duplicate so you have reproducibility) labe
l mRNA from 2 different tissues using 2 different
fluorescent dyes hybridize labelled mRNA to the
array wash analyze amount of color
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Functional Genomics
green mRNA 1 red mRNA 2 yellow overlap
of red and green intensities of each color
represent the amount of that mRNA in the
inital samples each spot represents a
different mRNA this picture represents 800
northern blots!!!