Professional Documents
Culture Documents
Part I
Why?
Understand the way in which plants and
animals, including humans, develop,
function and evolve
Investigate the molecular basis of disease
Develop products for medicine and crops
for agriculture
Solve crimes and paternity disputes
Investigate endangered species for
conservation management
Studying DNA
A number of methods have been developed that
can be used to identify the DNA (genetic) profile
of an individual
These methods can also be employed to
measure genetic differences between individuals
in a population
Techniques for working with DNA can be broken
down into four major categories:
Copying DNA
Cutting and pasting DNA
Measuring DNA length
Probing DNA
Extracting DNA
Extracting DNA
Break open (lyse) the cells or virus containing the DNA of interest.
This is often done by sonicating or bead beating the sample.
Vortexing with phenol (sometimes heated) is often effective for
breaking down protienacious cellular walls or viral capsids. The
addition of a detergent such as SDS is often necessary to remove
lipid membranes.
DNA associated proteins, as well as other cellular proteins, may be
degraded with the addition of a protease. Precipitation of the protein
is aided by the addition of a salt such as ammonium or sodium
acetate. When the sample is vortexed with phenol-chloroform and
centrifuged the proteins will remain in the organic phase and can be
drawn off carefully. The DNA will be found at the interface between
the two phases.
DNA is the precipitated by mixing with cold ethanol or isopropanol
and then centrifuging. The DNA is insoluble in the alcohol and will
come out of solution, and the alcohol serves as a wash to remove the
salt previously added.
Wash the resultant DNA pellet with cold alcohol again and centrifuge
for retrieval of the pellet.
After pouring the alcohol off the pellet and drying, the DNA can be resuspended in a buffer such as Tris or TE.
Copying DNA
How it works:
Separate the two strands with low heat
Add some base pairs, primer sequences, and
DNA Polymerase
Creates double stranded DNA from a single
strand.
Primer sequences create a seed from which
double stranded DNA grows.
Now you have two copies.
Repeat. Amount of DNA grows exponentially.
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Step 2 Annealing
Temperature decreased (50-60oC) in order for primers to anneal and
provide a starting point for DNA polymerase
Step 3 Extension
Temperature increased (72oC) which allows Taq polymerase to extend
DNA
Taq polymerase is a heat resistant DNA polymerase isolated from the
bacterium Thermus aquaticus which is found in hot springs and
hydrothermal vents
Denaturation
Raise temperature to 94oC
to separate the duplex form
of DNA into single strands
Design primers
To perform PCR, a 10-20bp sequence on either
side of the sequence to be amplified must be known
because DNA polymerase requires a primer to
synthesize a new strand of DNA
Annealing
Anneal primers at 50-65oC
Annealing
Anneal primers at 50-65oC
Extension
Extend primers: raise temp to 72oC, allowing Taq
pol to attach at each priming site and extend a
new DNA strand
Extension
Extend primers: raise temp to 72oC, allowing Taq
pol to attach at each priming site and extend a
new DNA strand
Repeat
Repeat the Denature, Anneal, Extension steps at their
respective temperatures
RT-PCR
PCR begins
here
Vector DNA
Restriction Enzymes
Discovered in the early 1970s
Used as a defense mechanism by bacteria to
break down the DNA of attacking viruses.
They cut the DNA into small fragments.
Can also be used to cut the DNA of organisms.
This allows the DNA sequence to be in a more
manageable bite-size pieces.
It is then possible using standard purification
techniques to single out certain fragments and
duplicate them to macroscopic quantities.
Restriction Enzymes
Definition:
A restriction enzyme is a bacterial enzyme that
recognises a short sequence of bases in a DNA molecule
and cuts the DNA at this recognition site.
The position where a cutting enzyme can snip is its
recognition sequence and is where a particular order of
nucleotides occurs.
Some restriction enzymes cut the two strands of a DNA
molecule at points directly opposite each other to produce cut
ends that are blunt.
Other cutting enzymes cut one strand at one point, but cut the
second strand at a point that is not directly opposite.
The overhanging cut ends made by these cutting enzymes are
called sticky. These sticky ends are complementary.
Pasting DNA
Once separated, DNA
fragments from different
sources can be joined
(ligated) together.
Sticky ended fragments will
initially join by hybridization or
complementary base pairing.
Bonds within the single
strands of DNA are then
repaired by DNA ligase (this is
similar to the action of DNA
ligase in linking of Ozaki
fragments on the lagging
strand during DNA replication)
Gel Electrophoresis
A copolymer of mannose and galactose, agaraose,
when melted and recooled, forms a gel with pores sizes
dependent upon the concentration of agarose.
The phosphate backbone of DNA is highly negatively
charged, therefore DNA will migrate in an electric field.
Gel Electrophoresis
DNA sequencing reactions are just like the PCR reactions for replicating DNA
with the exception that the reactions are run in the presence of a
dideoxynucleotide.
Sequencing reactions are set up in groups of four, e.g. one containing dideoxyA, one containing dideoxy-C, one containing dideoxy-G and one containing
dideoxy-T. Each reaction tube contains a mix of normal nucleotides (A,C,G, T)
and a small amount of the particular dideoxynucleotide.
Sooner or later ALL of the copies will get terminated by a T, but each time the
enzyme makes a new strand, the place it gets stopped will be random. In
millions of starts, there will be strands stopping at every possible T along the
way.
ALL of the strands we make started at one exact position. ALL of them end
with a T. There are billions of them ... many millions at each possible T
position. To find out where all the T's are in our newly synthesized strand, all
we have to do is find out the sizes of all the terminated products!
Assembling Genomes
Based on sequencing data, we can take fragments and put
them back together.
Not as easy as it sounds!!!!!
SCS Problem (Shortest Common Superstring)
Some of the fragments will overlap
We try to fit overlapping sequences together to get the shortest
possible sequence that includes all fragment sequences
Problems that may arise during this process:
DNA fragments contain sequencing errors
There are two complements of DNA we need to take into account both
directions of DNA
The repeat problem - 50% of human DNA is just repeats. If you have
repeating DNA, how do you know where it goes?
Analyzing a Genome
How to analyze a genome in four easy steps.
Cut it
Use enzymes to cut the DNA in to small fragments.
Copy it
Copy it many times to make it easier to see and detect.
Read it
Use special chemical techniques to read the small fragments.
Assemble it
Take all the fragments and put them back together. This is
hard!!!
Probing DNA
Nucleotide Hybridization
Create a Hybridization
Reaction
1.
2.
TAGGC T G
C
G
CT
A
T
ATCCGACAATGACGCC
4.
ACTGC
ACTGC
ATCCGACAATGACGCC
Great Homology
ACTGC
ATCCGACAATGACGCC
ATTCC
ATCCGACAATGACGCC
Less Homology
ACCCC
ATCCGACAATGACGCC
Low Homology
Southern Blotting
DNA Microarray
Affymetrix
DNA Microarray
DNA Microarrays
An array works by exploiting the ability of a given mRNA
molecule to hybridize to the DNA template.
Using an array containing many DNA samples in an
experiment, the expression levels of hundreds or thousands
genes within a cell by measuring the amount of mRNA bound
to each site on the array.
With the aid of a computer, the amount of mRNA bound to
the spots on the microarray is precisely measured,
generating a profile of gene expression in the cell.
An experiment on a microarray
In this schematic:
GREEN represents Control DNA
RED represents Sample DNA
YELLOW represents a combination of Control and Sample DNA
BLACK represents areas where neither the Control nor Sample DNA
Each color in an array represents either healthy (control) or diseased (sample) tissue.
The location and intensity of a color tell us whether the gene, or mutation, is present in
the control and/or sample DNA.
10
Forensics:
DNA technology in action
Forensic Identification
Identification using DNA is a powerful tool that can be
applied in many situations including:
forensic applications
Can the DNA found at a crime scene be matched to a person on the
national DNA database?
Is this blood spot from the victim or from the possible assailant?
In a rape case, is this semen from a previously convicted rapist?
Note that the allele frequencies vary within a population, with allele 11 being far
more common than allele 15.
Note also that the frequencies vary between populations, with allele 7 being about 20
times more common in Asian populations than in the other two populations.
For simplicity, STR loci that start with the letter D are identified by their chromosomal location
only, for example, D13 or D7. In reality, the naming of STRs is more complex because there are
multiple STR regions on the one chromosome and these two STRs (D13 and D7) are formally
identified as D13S317 and D7S820.
STR Profiling
To produce a DNA profile, multiple copies of the alleles at these nine STRs
are simultaneously produced using the polymerase chain reaction and the
various alleles are then separated and made visible with fluorescent dyes.
The resulting DNA profile is a series of coloured peaks at different locations,
with each peak being one allele of one specific STR. The location of each
peak indicates the size of the allele and hence the number of repeats.
Where sizes overlap, alleles of different STRs are distinguished by
fluorescent labels of different colours.
STR Profiling
[n x (n + 1)]/2.
8.5 billion to 1
Odds of blood on glove not being from R. Goldman, N. Brown-Simpson, and O.J. Simpson:
21.5 billion to 1
6.1 billion
2.8 million to 1
76 million to 1
Odds of getting killed driving to the gas station to buy a lottery ticket
4.5 million to 1
85 million to 1
85 million to 1
10 trillion to 1