Chapter 20 - Biotechnology

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CHAPTER 20
DNA TECHNOLOGY AND
GENOMICS
Insulin in the seeds
of this Safflower
plant
Insulin in themilk
of this Argentinian
cow!
DIABETES
Insufficient
insulin (gene
product) in the
body
“Over hot pastrami and corned
beef sandwiches, Herbert Boyer
and Stanley Cohen opened the
door to genetic engineering and
laid the foundations for gene
therapy and the biotechnology
industry.”
1996 Lemelson-MIT Prize
Winners
DNA technology – using DNA as a tool for
advancement in research and medicine
o Biotechnology – using “life” to make
useful products
• Recombinant DNA -genes from two
different sources - are combined in vitro into
the same molecule.
• Genetic engineering - the direct
manipulation of genes for practical
purposes.
Insulin
-a hormone
produced by pancreas
--a protein
---a gene product
-Diabetes Treatments:
-Supply artificial insulin
-Then insulin (a gene product)
has to be manufactured (how?)
-Gene therapy to fix defect
Goals of Bio/DNA technology
1) Isolate insulin or ‘gene of interest’ from the human genome
2) Engineer a recombinant DNA (ex. chimeric plasmid) that
has the ‘gene of interest’
3) Clone the recombinant DNA (ex: in bacteria) to make
million of copies
4) Now the gene of interest can be excised out of the cloned
bacteria colonies using probes that bind selectively to the
gene of interest (now you have millions of copies of your
gene)
5) You can introduce this gene into an egg/sperm/balstocyst
(early embryo) of any organism, or culture it to make your
human gene’s protein
Goal 1: Isolate gene of interest - ‘cut it’ out of human genome
Goal 2: Make recombinant DNA - ‘paste’ human gene in plasmid
• Restriction Enzymes:
found in bacteria
naturally -used to cut
foreign DNA in bacteria
• Most restrictions
enzymes are very
specific, recognize
short DNA nucleotide
sequences and cut at
specific point in these
sequences.
– Bacteria protect their own
DNA by methylation.
• Restriction Enzymes:
• Cut at Restriction Sites
on DNA
• Restriction sites are
often palindromes
• Restriction enzymes
leave sticky ends on the
DNA they cut
• Paste - seal with DNA
ligase
• How can you use this
information to make
recombinant DNA?
• Making Recombinant
DNA
• Cut at SAME Restriction
Sites on plasmid DNA
and DONOR DNA (with
gene of interest)
• Use DNA Ligase to join
the sticky ends
Goal 3: Clone the recombinant
DNA - reinsert the plasmid
(vector) back into bacteria and
let bacteria grow
• Step 1: Bacterial
transformation - gets
recombinant plasmid
DNA into bacteria (use
heat shock + CaCl2 to drill
holes so foreign DNA is
accepted)
• Now the recombinant
DNA can be “cloned” this
means - make many,
many copies of the DNA
by growing the bacteria
on agar plates
Vector - used to clone the recombinant DNA
Step 2a: How will you know
if the bacterial clone has
the recombinant DNA?
Luc+
Ori
Amp+
Plasmid Vector has:
Antibiotic (amp+/tet+)
resistance gene - grow the
bacterial culture in
antibiotics and select
colony that has resistance
Restriction sites
Ori Site -starts replication
Marker/reporter that can
be used to identify
recombinant event (Ex.
luciferase+ glowing gene
from FIREFLY DNA)
AP Lab
Negative Control Negative Control
Medium LB = Only Agar LB + Amp
No Amp
Genes
No luc+
No luc+
LB+ Amp
luc+
Luc+
Amp
Amp
Amp
Amp is an antibiotic; Amp+ is a gene
on the plasmid that confers resistance.
Luc+ makes bacteria glow.
Another Selection technique to
pull out colony with gene of
interest:Step 2b
-Nucleic acid hybridization
depends on base pairing
between DNA/RNA of interest
and a complementary
sequence, a nucleic acid
probe
-A radioactive or fluorescent tag
labels the probe.
- Bacterial colonies are
transferred to a special filter
paper (nitrocellulose)
- Colony containing the gene of
interest can be identified using
a probe that hybridizes (pairs)
with the gene of interest
• Can you take a human gene and insert it
into a bacteria using a plasmid vector and
problems
making recombinant DNA
expect it2to
make in
protein?
in eukaryotes:
• AHEM! What about introns? (remember a
How the
do you
get theand
correct
mRNA
gene has1)both
introns
exons!)
out of a human cell (to make the
• Solution: Use
the spliced mRNA. Use
cDNA)
reverse transcriptase and synthesize a
In other words – how can the correct
complementary
strand.
RNA/DNA be recognized in a cell?
– This is complementary DNA (cDNA),
Howtodo
open
a eukaryotic cell
– Attach 2)
cDNA
a you
vector
forupreplication,
and and
maketranslation
it take up foreign
transcription,
inside DNA?
bacteria.
• Several techniques facilitate entry of foreign DNA
in Eukaryotic Cells.
– Electroporation- brief electrical pulses create a
temporary hole in the plasma membrane
– Inject DNA into individual cells using microscopically thin
needles.
– DNA is attached to microscopic metal particles and fired
into cells with a gun.
So, can you store all the human
genes in a recombinant form?
• Book = bacteria
• Each Chapter =
Different Plasmid
• What will the
human gene be?
• Passages in
chapters = human
gene of interest
• Genomic Library –
stores all the genes
of an organisms in
a library of vectors
• cDNA library mRNAs are
converted to
complimentary or
cDNA and stored in
the library
Cloned genes are stored in DNA
libraries
• “Shotgun” cloning - a mixture of fragments
from the entire genome is included in
thousands of different recombinant plasmids.
Human genome project
1990 approach:
Use existing linkage maps
and break up chromosomes
into overlapping parts;
make clones of the DNA assigned to scienitists all
over the country
Determine the sequence of
each individual part and
find the order of 300 billion
base pairs!
Celera- Shotgun approach:
Chop up the entire genome
into small fragments
Determine the sequence of
each individual part and
then overlap segments to
find the order of 300 billion
base pairs!
Diploid Human DNA
sequence determined in
2007 was Craig Venter’s!
• In addition to plasmids, bacteriophages can
be cloning vectors for making libraries.
Do you always need a
plasmid/vector to clone DNA?
• The polymerase
chain reaction
(PCR) can clone
DNA without
using a vector
• HOW????
• WHAT DOES
THIS IMPLY?
• PCR-Reagents
• 1) Primers - 20
nucleotide long single
stranded DNA
sequences that can
hybridize with the gene
of interest DNA at its
very end.
• 2) Free nucleotides
• 3) Special DNA
polymerase called Taq
Polymerase - from
bacteria that live in hot
springs
Fig. 20.7
• PCR-Steps
• 1) Heat to 95c - denature DNA
to break up ds DNA in gene of
interest
• 2) Lower the temp. to 60c Primers can bind to the ends of
gene of interest
• 3) Next increase temp to 75c for
Taq Polymerase to work well - it
adds free nucleotides to 3’ end
of primers
• 4) One cycle - 2 strands, 2
cycles - 4 copies, 3 cycles - 8
copies, …. 25 cycle - 33 million
copies!
-Amplify a small amount of DNA
quickly!
• Why PCR, when plasmids are available re-inventing the wheel, you say?
• PCR can make billions of copies of a targeted
DNA segment in a few hours.
• How can it do that?
• PCR, is a three-step cycle: heating, cooling, and
replication
• PCR, is like an atomic bomb explosion - HOW?
• -a chain reaction that produces an exponentially
growing population of DNA molecules
• Remember- PCR uses an UNUSUAL DNA
polymerase, isolated from bacteria living in hot
springs, (can withstand the heat needed to separate
the DNA strands at the start of each cycle).
Goal
Get 4:
DNA
Remove
Fragments
gene of
(use
interest from the cloned library using gel
electrophoresis
Restriction Enzyme digest))
Agarose
• Comparison of 2 genomes: Gel Electrophoresis Gel
• DNA is –ve; Pass
electric
Pores in
gel thru current
which
DNAelectrode
• DNA will move
to +ve
fragments move
• So what?
Know this! Rate of movement of DNA Fragment mainly
depends on length or number of base pairs
Long fragment
Short fragment
Apply an electric
current - DNA
moves to +ve pole
Particle
DNA Gel electrophoresis
• Can detect differences in DNA
fragment size/length
• DNA fragments arise from
restriction enzymes that cut
DNA into different sizes
• Depending on where the ‘cutting’
or restriction sites lie on the
noncoding regions of the
chromosome you get different
DNA gel patterns - so every
person has a unique restriction
digest profile - RFLP (rif- lips)
Restriction Fragment Length
Polymorphism (many forms)
• You inherit these restriction sites
according to Mendelian rules
DNA FINGERPRINTING
DNA FINGERPRINTING USING RFLPS
• Restriction fragment analysis is sensitive enough
to distinguish between two alleles of a gene that
differ by only base pair in a restriction site.
Remember these differences
are in the noncoding parts but restriction sites may lie
close to a gene and serve as a
‘marker’ for that gene
Fig. 20.9
• For our three individuals, the results of these steps
show that individual III has a different restriction
pattern than individuals I or II.
Summary: Restriction fragment length
polymorphisms (RFLPs) – we each have a different
site where the restriction enzymes cut - DNA
fingerprinting maps this out on a gel
• RFLPs can serve as a genetic marker for diseases
Fig. 20.10
Southern blotting
• Helps identify the gene of interest in the gel
electrophoresis bands
• Uses probes to hybridize with the gel band
copy on a nitrocellulose paper
• The band of interest can be cut out and the
gene of interest can be isolated - now we
have millions of coies of pure gene of
interest.
Insert the gene of interest in
an egg/sperm and create a
transgenic animal
• Microinject the gene of interest into the
egg/sperm nucleus!
• Animal makes human protein!
• Plant is totipotent - so no need to use
embryos - get it into leaf or stem or root of a
plant and culture it - these cells can make a
transgenic embryo!
Microarrays: Attach all the dna/genes from an
organism as single strands on a microarray
plate. Ad cDNA from a patient .Spots where
any of the cDNA hybridizes fluoresce with an
intensity indicating the relative amount of the
mRNA that was in the tissue.
Gene chips
• In chromosome
walking, the
researcher starts
with a known
DNA segment
(cloned,
mapped, and
sequenced) and
“walks” along the
DNA from that
locus, producing
a map of
overlapping
fragments.
Fig. 20.11
Genome sequences provide clues to important biological questions
• Genomics, the study of genomes based on their DNA sequences, is
yielding new insights into fundamental questions about genome
organization, the control of gene expression, growth and development,
and evolution.
• Rather than inferring genotype from phenotype like classical geneticists,
molecular geneticists try to determine the impact on the phenotype of
details of the genotype.
•
By doing more mixing and matching of modular elements, humans - and
vertebrates in general - reach more complexity than flies or worms.
– The typical human gene probably specifies at least two or three different
polypeptides by using different combinations of exons.
• Along with this is additional polypeptide diversity via post-translational
processing.
– The human sequence suggests that our polypeptides tend to be more
complicated than those of invertebrates.
• While humans do not seem to have more types of domains, the
domains are put together in many more combinations.
•
About half of the human genes were already known before the Human
Genome Project.
To determine what the others are and what they may do, scientists compare
the sequences of new gene candidates with those of known genes.
– In some cases, the sequence of a new gene candidate will be similar in
part with that of known gene, suggesting similar function.
– In other cases, the new sequences will be similar to a sequence
encountered before, but of unknown function.
– In still other cases, the sequence is entirely unlike anything ever seen
before.
• About 30% of the E. coli genes are new to us.
•
•
Comparisons of genome sequences confirm very strongly the evolutionary
connections between even distantly related organisms and the relevance of
research on simpler organisms to our understanding of human biology.
– For example, yeast has a number of genes close enough to the human
versions that they can substitute for them in a human cell.
– Researchers may determine what a human disease gene does by
studying its normal counterpart in yeast.
– Bacterial sequences reveal unsuspected metabolic pathways that may
have industrial or medical uses.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• Studies of genomes have also revealed how
genes act together to produce a functioning
organism through an unusually complex network
of interactions among genes and their products.
• To determine which genes are transcribed under
different situations, researchers isolate mRNA
from particular cells and use the mRNA as
templates to build a cDNA library.
• This cDNA can be compared to other collections of
DNA by hybridization.
– This will reveal which genes are active at different
developmental stages, in different tissues, or in tissues
in different states of health.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• Perhaps the most interesting genes
discovered in genome sequencing and
expression studies are those whose
function is completely mysterious.
• One way to determine their function is to
disable the gene and hope that the
consequences provide clues to the gene’s
normal function.
– Using in vitro mutagenesis, specific changes
are introduced into a cloned gene, altering or
destroying its function.
– When the mutated gene is returned to the cell,
it may be possible to determine the function of
the normal gene by examining the phenotype
of the mutant.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• In nonmammalian organisms, a simpler
and faster method, RNA interference
(RNAi), has been applied to silence the
expression of selected genes.
– This method uses synthetic double-stranded
RNA molecules matching the sequences of a
particular gene to trigger breakdown of the
gene’s mRNA.
– The mechanism underlying RNAi is still
unknown.
– Scientists have only recently achieved some
success in using the method to silence genes
in mammalian cells.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• The next step after mapping and sequencing
genomes is proteomics, the systematic study of
full protein sets (proteomes) encoded by
genomes.
– One challenge is the sheer number of proteins in
humans and our close relatives because of alternative
RNA splicing and post-translational modifications.
– Collecting all the proteins will be difficult because a
cell’s proteins differ with cell type and its state.
– In addition, unlike DNA, proteins are extremely varied in
structure and chemical and physical properties.
– Because proteins are the molecules that actually carry
out cell activities, we must study them to learn how cells
and organisms function.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• Genomic and proteomics are giving biologists an
increasingly global perspective on the study of life.
• Eric Lander and Robert Weinberg predict that
complete catalogs of genes and proteins will
change the discipline of biology dramatically.
– “For the first time in a century, reductionists [are yielding]
ground to those trying to gain a holistic view of cells and
tissues.”
• Advances in bioinformatics, the application of
computer science and mathematics to genetic and
other biological information, will play a crucial role
in dealing with the enormous mass of data.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• These analyses will provide understanding of the
spectrum of genetic variation in humans.
– Because we are all probably descended from a small
population living in Africa 150,000 to 200,000 years ago,
the amount of DNA variation in humans is small.
– Most of our diversity is in the form of single nucleotide
polymorphisms (SNPs), single base-pair variations.
• In humans, SNPs occur about once in 1,000 bases, meaning
that any two humans are 99.9% identical.
– The locations of the human SNP sites will provide useful
markers for studying human evolution and for identifying
disease genes and genes that influence our
susceptibility to diseases, toxins or drugs.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
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