Download DNA

Working with DNA:
Isolation and Fingerprinting
Funding and support received
from…
Today’s Agenda:
1) Introduction
2) Safety
3) Basic Practice “Using a Pipetteman”
4) DNA Isolation Procedures
5) Restriction Enzymes and Gels
6) Yellowstone National Park and Bacterial Mats
7) Practice DNA Fingerprinting Problems
8) Analysis of our Fingerprinting Gels
9) Bacteria and DNA Basics
10)Closing
Our Research Project - What
We Are Cloning and Why
• We hope to identify
•
new hot spring
bacteria that cannot
be grown on lab
media
To study these
organisms, we extract
DNA from hot springs
that contain unknown
bacteria
Our Research Project - What
We Are Cloning and Why
• We clone a specific
•
•
identification gene (the
16S gene) from the hot
spring DNA
We place each hot spring
gene into E. coli, our
cloning factory
And then we fingerprint
and DNA sequence each
hot spring clone
Words to the cautious…
Neither the E. coli we use nor the
hot spring bacteria we study have
ever been shown to be pathogenic.
Although you will be working with E.
coli, you will never come in contact
with hot spring bacteria… just their
DNA after it has been extracted
from the once-living cells.
Introduction:
• All living things contain cells
• Eukaryotes: more than one cell
• Prokaryotes: one cell organisms
The Boring (Yawn!!) Eukaryotic
Plant and Animal Cells…
The Exciting Bacterial Cell…
Bacteria come in many different
shapes and sizes…take a quick
look…
Bacteria can replicate easily…
• To grow, bacteria divide
and
divide and divide again.
• Problem: If you started with only 1
bacterial cell, and it divided 10 times,
how many bacteria would you then
have??
Bacteria are everywhere…
Don’t panic!!
This is a good thing.
We have bacteria growing on our
bodies which are supposed to be
there.
What are Bacteria?
• Bacteria are prokaryotes, meaning they are only
one celled organisms. They are very small and
can be harmful or beneficial.
Bacteria can cause diseases, like
we all know…
Bacteria can also have beneficial
uses…
Bacterial Cell Components…
Plasmids can also be found in
bacterial cells:
• Plasmids are: Mini•
•
•
•
chromosomes found only
in some bacteria
(1,000-10,000 base pairs)
Free-floating in the
cytoplasm - not
membrane-bound like
chromosome
Naturally carry many
antibiotic resistance
genes
Replicate on their own
Plasmids and Cloning
• Bacteria are used in genetic engineering
and cloning because they serve as the
factories for expressing foreign genes like
insulin. Without plasmids, there would be
no way to clone and express foreign
genes.
Now we are going to do some
work!!!
DNA Precipitation
What are we using now?
• 3M Sodium acetate: contributes ions to
bind with positive phosphates open on
DNA
• Isopropanol: polar solution which
attaches to DNA for precipitation
• - 80C Freezer: Speeds the precipitation
reaction with low amounts of DNA
DNA…
the code of life
What do we know about DNA?
• Structure:
Composed of nucleotides
(monomer) consisting of:
1) phosphate group
2) deoxyribose sugar
3) one of four nitrogen bases
What do we know about DNA?
• Structure:
Nitrogen bases are named:
- adenine (A)
- guanine (G)
- thymine (T)
- cytosine (C)
What do we know about DNA?
• Structure:
• The structure of these
nucleotides determines
how they fit together.
• Adenine fits with Thymine
• Guanine fits with Cytosine
What do we know about DNA?
• Structure:
• DNA is “double-stranded”
• The nucleotides are
•
•
linked together covalently
Phosphate – Sugar –
Phosphate – Sugar etc.
This is the “backbone”
What do we know about DNA?
• Structure:
• The two strands are
oriented in opposite
directions
• The two strands are
wound around each other
forming the “helix”
structure
What do we know about DNA?
• Function:
• Codes for 80,000 genes,
which form proteins…the
building blocks of life.
Eukaryotic Deoxyribonucleic Acid
• DNA for Short
• Double helix - two strands made up of
A, T, G, and C bases
• Complex organisms - many linear
chromosomes (10,000,000,000 or more
base pairs)
Plant or Animal DNA Strand:
Prokaryotic Deoxyribonucleic Acid
• Bacteria - one circular chromosome
(1,000,000 base pairs)
• Chromosomes, in both cases, are held by
proteins to the cell or nuclear membrane
• Most RNA is translated into proteins that have
structural or functional jobs in cells
Bacterial DNA Strand
Let’s get our samples now and
continue on with our isolation…
• Centrifuge: Spins solution at high speed
to concentrate DNA at the bottom
• TE: buffer at pH 8.0
• RNAse: enzyme which removes RNA
present in sample through digestion
Restriction Enzymes and Gels
Restriction Enzymes
• Cut specific sequences of
DNA
• Many different kinds
• Named after organism
they came from, enzyme
number
• E.g. EcoR1
Bacteria Produce Restriction
Enzymes
• Uniquely bacterial
protection
mechanism…why?
• Restriction enzymes
are short nucleotide
sequences isolated
from bacteria cells
that protect them
from virus.
Bacteria Produce Restriction
Enzymes
• When a viral DNA enters
the bacterial cell, the
restriction enzyme is able
to recognize a specific
sequence (restriction site)
on the DNA molecule,
which is usually 4-8
nucleotides long. The
restriction enzyme will cut
the viral DNA at these
sites and hence restrict
the growth of the virus.
Bacteria Produce Restriction
Enzymes
• Several hundreds of these
enzymes have been
isolated from various
organisms and most are
available commercially.
These enzymes are used
to cut a segment of gene
from a human DNA
molecule.
DNA Fingerprinting
DNA Fingerprinting
• DNA fragments are
separated using gel
electrophoresis
• Each band represents
the DNA which has
been cut into smaller
pieces using
restriction enzymes
Gel Electrophoresis
From your studies of DNA, can
you tell me what charge DNA has?
Gel Electrophoresis
• Gel is made of water
and agarose
• Wells on one end are
where gels will be
loaded with our
samples
Gel Electrophoresis
• The gel box contains
water and buffer to keep
the pH constant
• Gel box has platinum wire
that conducts protons and
electrons
• Gel box will be wired to
the power source
following the load
Gel Electrophoresis
• To the strand of DNA
moving through the
agarose, the gel looks
like a big mesh-like
maze
• The DNA travels
through the maze as
fast as it’s size will
allow
Gel Electrophoresis
• DNA moves from the
negative towards the
positive
• Smaller – faster
• Larger – slower
• Where will these
three end?
Gel Electrophoresis
• Review:
** DNA travels – to +
** When the power
supply turns off,
we can see where
the bands are and
infer which are
bigger and smaller
** Small goes far
** Large goes not far
DNA Fingerprinting Questions
and Answers
Do you know the answers to
these questions?
DNA Fingerprinting
• How good (accurate) is it at
identification. For example, is it as
good as classical fingerprints?
Question 1:
• How good (accurate) is it at
identification. For example, is it as
good as classical fingerprints?
• Answer: In theory, with the exception of
identical twins, EVERYONE on this planet
has a different DNA fingerprint. That is,
DNA fingerprinting IS as good
(distinctive/unique/specific) as classical
fingerprinting for identification.
Question 2:
• What are its advantages?
• Answer: In theory DNA fingerprinting will work with
much smaller amounts of material than a classical
fingerprint & DNA lasts much longer than classical
fingerprints. DNA-containing samples that are many
years old (up to 25 million yr.) are still usable. Only very
tiny quantities of DNA are required in order to carry out
a highly accurate test. For example, dried blood, semen,
spit, skin etc. on samples stored in dusty files for years
are still usable. Samples of mixed DNA's can also be
used. DNA containing evidence is much harder to clean
up at a crime scene than other evidence, like classical
fingerprints.
Question 3:
• What are its limitations?
• Answer: There currently are no accepted Federal
standards for controlling the quality of DNA testing
nationwide. Poor quality & poorly controlled testing can
lead to QUESTIONABLE and SHODDY RESULTS.
• Even if there is a perfect match between DNA, you can
not say HOW the DNA containing sample got there or
WHEN. In the O.J. trial a VALID question was raised
about the possibility of evidence being planted. What
makes this charge so powerful is the EXTREME
SENSITIVITY of the procedure.
Now, how do we come up
with those different bands?
Answer: Restriction Enzymes
Let’s do an example of DNA
Fingerprinting together…
DNA Fingerprinting Example:
• Two men fitting the description of a
robber were caught in the vicinity of the
crime. Both had cuts on their arms which
they “explained away.” DNA samples were
taken from each suspect and from the
broken window at the scene of the crime.
DNA Fingerprinting Example:
• Using DNA Fingerprinting and Restriction
Enzymes, we can determine which of the
men was the robber!!
• We can cut each sample (one from each
suspect and one from the crime scene)
with two different enzymes, run them on a
gel and compare the results
So, how do we organize what we
know? We organize the gel lanes…
Lane
Description:
1
DNA sample from crime scene cut w/ Enzyme 1
2
DNA sample from crime scene cut w/ Enzyme 2
3
DNA sample from Suspect 1 cut with Enzyme 1
4
DNA sample from Suspect 1 cut with Enzyme 2
5
DNA sample from Suspect 2 cut with Enzyme 1
6
DNA sample from Suspect 2 cut with Enzyme 2
Fingerprinting Gel Projected
Results:
Practical applications of
DNA technology
Practical applications of DNA
technology
• Diagnosis of diseases
includes:
–
–
–
–
Huntington’s
PKU
cystic fibrosis
Duchenne’s muscular
dystrophy
Practical applications of DNA
technology
• Human gene therapy
• Somatic cell therapy
versus germ cell
therapy
Practical applications of DNA
technology
• Pharmaceutical
products:
Insulin
human growth
hormone
• Protection from viral
infection
Practical applications of DNA
technology
Forensic uses
DNA fingerprinting
RFLPs and simple
tandem repeats
(microsatellite DNA
repeats of different
lengths)
Practical applications of DNA
technology
• Environmental uses:
• Genetically engineered
microbes for mining,
cleaning up toxic
wastes, etc.
Practical applications of DNA
technology
Agricultural uses
• Animal husbandry
– Transgenic animals
– Gene knock-in or knockout animals (requires
homologous
recombination)
– Cloned animals
• Genetic engineering in
plants
– Can grow many plants
from a single cell