DNA Technology and Genomics

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

Working with DNA

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

Why we need so many copies

Biologists needed to find a way to read DNA codes.
How do you read base pairs that are angstroms in
size?
It is not possible to directly look at it due to DNAs
small size.
Need to use chemical techniques to detect what you
are looking for.
To read something so small, you need a lot of it, so
that you can actually detect the chemistry.

Need a way to make many copies of the base

pairs, and a method for reading the pairs.

Polymerase Chain Reaction

(PCR)
Polymerase Chain Reaction (PCR)
Used to massively replicate DNA sequences.
Exploits enzymes and process of replication
that normally occurs in cells.

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.
1248163264128256

Polymerase Chain Reaction

Problem: Modern
instrumentation cannot
easily detect single
molecules of DNA, making
amplification a prerequisite
for further analysis
Solution: PCR doubles the
number of DNA fragments
at every iteration

Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) is broken down into three
separate steps which are repeated until enough DNA is obtained
(usually between 25 and 40 cycles)
Step 1 Denaturation
Temperature is raised (94oC) in order to separate dsDNA into single
strands

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

Polymerase Chain Reaction

RT-PCR: Variation of PCR

PCR reaction amplifies DNA from a single copy in the absence of

cells
RT-PCR is a variant of PCR in which DNA is first reverse
transcribed (copied in reverse) from RNA extracted from cells prior
to being amplified by PCR as per usual protocol
Reverse transcription process involves the use of a reverse
transcriptase enzyme and various other reagents including either a
primer for a specific gene or an oligo-dT primer (string of Ts that will
bind to poly-A tail of RNA.
Enzyme, reagent mix, primer and RNA are usually incubated at
37oC for 1 hour before the RT enzyme is inactivated at 72oC for
approximately 15 minutes. The resulting cDNA is then used as the
template for a PCR reaction.

RT-PCR

PCR begins
here

Cloning DNA to achieve copies

Use restriction enzymes and DNA ligase to insert the

fragment of interest into the genome of another organism
(e.g. bacteria) in order for it to multiply.
The resulting DNA is referred to as recombinant DNA as
the genes from two different organisms are combined.
Once you have a large quantity of bacteria, you will be
able to isolate a large quantity of the gene of interest

Vector DNA

Cutting and Pasting 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.

Blunt and Sticky Ends

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)

Measuring DNA Length

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

The size of DNA fragments can then be determined by comparing

their migration in the gel to known size standards
Ethidium bromide or other dyes that bind to DNA are added prior to
electrophoresis in order to visualize migration of DNA
Ethidium bromide flouresces bright orange when exposed to UV
light

Reading DNA DNA Sequencing

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.

Dideoxyribonucleotides are the same as nucleotides, with one exception. They

do not have 3' hydroxyl group, so once a dideoxynucleotide is added to the end
of a DNA strand, there's no way to continue elongating it.

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.

Taking dideoxy-C as an example, replication of DNA will occur as per a PCR

reaction. MOST of the time when a C' is required to make the new strand, the
enzyme will get a good one and there's no problem. MOST of the time after
adding a C, the enzyme will go ahead and add more nucleotides. However, 5%
of the time, the enzyme will get a dideoxy-C, and that strand can never again be
elongated. It eventually breaks away from the enzyme, a dead end product.

Reading DNA DNA Sequencing

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!

Reading DNA DNA Sequencing

Gel electrophoresis can be used to separate the fragments

by size and measure them.
The dideoxynucleotides present in the fragments have been
labelled with a radioisotope or a flourescent dye. In the case
of the latter, these can be read by a laser and the information
feed back to computer.
Following electrophoresis and visualization of fragments we
can determine the sequence. Smallest fragments are at the
bottom, largest at the top. The positions and spacing shows
the relative sizes. At the bottom are the smallest fragments
that have been terminated by dideoxynucleotides.

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!!!

Bioinformatics takes over

What can we learn from the sequenced DNA.
Compare interspecies and intraspecies.

Probing DNA

Nucleotide Hybridization

Single-stranded DNA or RNA will naturally bind to complementary

strands.
Hybridization is used to locate genes, regulate gene expression, and
determine the degree of similarity between DNA from different
sources.
Hybridization is also referred to as annealing or renaturation.
Hybridization uses oligonucleotides to find complementary DNA or
RNA seqments. Oligonucleotides are single-stranded DNA molecules
of 20-30 nucleotides in length.
Oligonucleotides are made with DNA synthesizers and tagged with a
radioactive isotope or fluorescent dye
Molecular techniques based on hybridization include Southern blotting,
Northern blotting and microarrays.

Create a Hybridization
Reaction
1.

2.

Hybridization is binding two genetic

sequences. The binding occurs
because of the hydrogen bonds [pink]
between base pairs.

When using hybridization, DNA must

first be denatured, usually by using
use heat or chemical.

TAGGC T G

C
G
CT
A
T

ATCCGACAATGACGCC

Create a Hybridization Reaction Cont.

3.

4.

Once DNA has been denatured, a

single-stranded radioactive probe [light
blue] can be used to see if the
denatured DNA contains a sequence
complementary to probe.

Sequences of varying homology stick

to the DNA even if the fit is poor.

ACTGC
ACTGC
ATCCGACAATGACGCC

Great Homology

ACTGC
ATCCGACAATGACGCC
ATTCC
ATCCGACAATGACGCC

Less Homology

ACCCC
ATCCGACAATGACGCC

Low Homology

Southern Blotting

Cut total genomic DNA with restriction

enzymes and separate by electrophoresis
Blot the fragments onto nitrocellulose filter
paper (the Southern blot)
Probe the blot for a particular DNA region
of interest using a specific labelled
oligonucleotide.
Wash blot to remove oligonucleotide that
has not bound.
Identify gene or region of interest by
visualizing regions where probe has
hybridised with DNA on Southern blot.
Northern blotting follows a similar process
to Southern blotting except that RNA is
run on the initial gel and the
oligonucleotide probes are used to detect
expression of particular genes.

DNA Microarray

Affymetrix

Microarray is a tool for

analyzing gene expression
that consists of a glass slide.
Each blue spot indicates the location of a PCR
product. On a real microarray, each spot is
about 100um in diameter.

DNA Microarray

Millions of DNA strands

build up on each location.

Tagged probes become hybridized

to the DNA chips 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

Each of us is genetically unique, with the exception of identical

(monozygotic) siblings. While phenotypic differences are apparent
among us, the most fundamental expression of our uniqueness is in
our genetic material, DNA.
Today, individuals can be identified through a technique known as
DNA profiling.
The amount of DNA needed for DNA profiling is very small because
DNA can be amplified through the polymerase chain reaction (PCR).
One persons DNA profile is constant, regardless of the type of cell
used to prepare the profile. A DNA profile prepared from a persons
white blood cells is identical to that prepared from the same
persons skin cells or other somatic cells.
Because DNA molecules are only slowly degraded, DNA profiling
can be carried out on biological samples from crime scenes from
years ago, and this profiling has led to the solution of many cold
cases worldwide.

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?

mass disasters, such as passenger aircraft crashes, the 9/11

terrorist attacks, the Bali bombings
Can the various remains that have been recovered be matched to a
particular person known to have been on-site?

identification of human remains

Are these remains those of a particular missing person?
Who was the unknown child, tagged as body number 4, recovered
after the sinking of the Titanic in 1912?

What DNA is used for identification

Depending on the purpose and circumstances of the
identification, the DNA used comes from either the
chromosomes (nuclear DNA) or from mitochondria
(mtDNA).
In both cases the identification depends on the existence
of segments of DNA that vary greatly between
individuals. Such regions of DNA are termed
hypervariable.
Hypervariable regions of DNA that are currently used for
identification are:
short tandem repeats (STRs) in the nuclear DNA, also known
as microsatellites.
hypervariable regions (HVRs) in the non-coding region of
mtDNA.

STRs and HVRs

Short Tandem Repeats (STRs) in the nuclear DNA, also known as microsatellites.
A large number of STRs are present on different human chromosomes.
DNA from STRs can identify one person uniquely (apart from identical siblings).
DNA samples from relatives are not required.
Used when there is a need to match a DNA sample from a crime scene to just one
particular person.
Hypervariable Regions (HVRs) in the non-coding region of mtDNA.
mtDNA identification is less precise because persons from the same maternal line
have identical mtDNA profiles.
mtDNA is used only when chromosomal DNA cannot be recovered or when
chromosomal DNA is degraded because of age.
Identification using mtDNA is mainly applied either
to identify victims of mass disasters where the names of the victims are known but
where identification of the remains by conventional means, such as visual inspection or
dental records, cannot be done, or
to identify decomposed remains when the identity is suspected to be one of a few
particular missing persons. In both cases, there must be living relatives on the maternal
line to provide mtDNA for comparison with the mtDNA from the remains.

DNA Fingerprinting - HVRs

The original technique of identification using DNA was

called DNA fingerprinting and was developed as an
identification tool in 1985 by Professor Sir Alec Jeffreys
This technique used DNA from hypervariable
regions, known as minisatellites, that are located near
the ends of chromosomes. Minisatellites are
chromosomal regions where sequences of 9 to 80
base pairs are repeated tens or hundreds of times.
DNA fingerprinting involved cutting minisatellites from
the chromosomal DNA with a restriction enzyme (Hin
fI), separating the DNA fragments by electrophoresis,
transferring them to a membrane using Southern
blotting and exposing the fragments to one probe that
hybridised to a base sequence present in all the
minisatellites.
This probe, known as a multi-locus probe, carried a
radioactive label. The final result seen on an
autoradiograph was a pattern of up to 36 bands,
something like a barcode, with each band being one
allele of one of the minisatellites. Because of the
variation between individuals, each DNA fingerprint is
unique.

The figure above shows the simplified

DNA fingerprints of two people based
on just four hypothetical minisatellites.
In actual DNA fingerprinting, the pattern
for each individual has many more
bands.

DNA profiling - STRs

DNA fingerprinting has been replaced by a technique known as DNA

profiling that uses short tandem repeats (STRs). These are
STRs are hypervariable regions of chromosomes where sequences
of just two to five base pairs are repeated over and over. These
regions are very common and hundreds are scattered throughout
the human chromosomes.
STRs are termed short because the repeat sequences are only 2 to
5 base pairs long, and tandem because the repeats occur one after
the other. However, the number of repeats at an STR locus can
vary between people and each variation is a distinct allele.
The number of repeats of a 4-base pair sequence at one STR locus
on the number-5 chromosome ranges from 7 to 15.
In most cases, the alleles at an STR locus on a human chromosome
are named according to the number of repeats and so are identified
as allele 7, allele 8 and so on. The figure below shows an STR with
7 repeats of the sequence CATT.

DNA profiling - STRs

At each STR locus, one individual is either homozygous or heterozygous

and so can have a maximum of just two different alleles. These alleles are
inherited in a Mendelian fashion.
The figure below shows that a person who is heterozygous 5/7 at one
particular STR locus has one allele with 5 repeats and another allele with 7
repeats.
Within the gene pool of a population, however, many different alleles can
exist at each STR locus.

Frequencies of the alleles at the D5 STR locus on the number-5

chromosome for three sample populations
in Australia.

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.

Why use STRs rather than

minisatellites?

Compared with DNA fingerprinting, DNA profiling:

is far more sensitive and requires smaller quantities of DNA (even a
pinhead sized spot of blood can provide sufficient DNA) and the STRs
can be amplified by the polymerase chain reaction (PCR)
is based on alleles whose sizes allow fragments differing by just one
base pair to be distinguished
is carried out in a much shorter time hours rather than days
uses several single-locus probes rather than one multi-locus probe
uses coloured fluorescent labels to visualise the STRs rather than
radioactive labels so that each different STR allele can be identified by
colour as well as by size
produces less complex patterns that are more easily interpreted

In addition, unlike minisatellites, population data on allele

frequencies of STR alleles can be obtained.

DNA Profiling in Australia

All Australian states use a common method of DNA
profiling for forensic purposes that involves nine STRs
from different human chromosomes.
These STR markers were chosen for this purpose
because they are reproducible and robust, easy to score,
are highly informative and have low mutation rates.
In addition, a tenth marker (that is not an STR) is used to
identify the gender of the individual. This gender marker
is the Amel locus that is present on both the X
chromosome and the Y chromosome.
The Amel gene on the X chromosome is just 107 base
pairs long while that on the Y chromosome contains 113
base pairs. As a result, the gender of a person can be
identified from this marker.

Loci currently used for DNA

profiling in Australia

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

A person shows either

one or two peaks at
each STR loci, where a
peak corresponds to an
allele, depending on
whether the individual is
homozygous or
heterozygous at that
locus.
For the Amel gender
marker, if just a single
peak with a size of 107
base pairs appears on
the profile, the person is
female; if two peaks are
detected, one at 107
and the second at 113
base pairs, then the
person is male.
This person is female.
They are heterozygous for loci D3, vWA, FGA, D18 and D7.
They are homozygous for loci D8, D21, D5 and D13.

Is STR Profiling reliable?

STR loci generate many different genotypes (profiles)

For one gene locus with n different alleles, the number of different
genotypes possible is:

[n x (n + 1)]/2.

An STR locus with 14 alleles can produce 105 different genotypes in a

population and a different STR locus with nine alleles can have 45 different
genotypes. Together, these two STR loci produce 105 45 = 4725 different
genotypes.
As the number of STR loci increases, the number of different genotypic
combinations in a population increases enormously.
As a result, a DNA profile based on nine STRs will be a unique combination
that allows a person to be identified with a very high level of probability.
The chance that the DNA profile of one person will be identical with that of
another person (except for an identical sibling) is one in many billions.

O.J. Simpson capital murder case,1/95-9/95

Odds of blood in Ford Bronco not being R. Goldmans:
6.5 billion to 1

Odds of blood on socks in bedroom not being N. Brown-Simpsons:

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

Number of people on planet earth:

6.1 billion

Odds of being struck by lightning in the U.S.:

2.8 million to 1

Odds of winning the Illinois Big Game lottery:

76 million to 1

Odds of getting killed driving to the gas station to buy a lottery ticket

4.5 million to 1

Odds of seeing 3 albino deer at the same time:

85 million to 1

Odds of having quintuplets:

85 million to 1

Odds of being struck by a meteorite:

10 trillion to 1