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Week 4
Methods and the Human Genome Project
What is PCR?
Polymerase chain reaction (PCR) is a common lab technique used to make millions or billions of copies of a particular region of DNA
This region can be anything the experimenter wants to know more about – it could be a gene with a function a researcher wants to understand
It is an in vitro practice
PCR requires a DNA polymerase enzyme that makes new strands of DNA, using the existing strands as templates – just like in DNA replication in an organism. In PCR, this DNA polymerase is usually Taq polymerase
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Taq is named after the heat-tolerant bacterium it was isolated from (thermus aquaticus)
Interestingly, Taq was first discovered at Yellowstone National Park
Thermus aquaticus is a thermophile bacterium. is stable at elevated temperatures.
DNA using Taq is achieved at the optimal temperature of 70°C and is used during PCR to denature the template DNA, or separate its strands
Taq polymerase can only make DNA if it’s given a primer – a short sequence of nucleotides that provides a starting point for DNA synthesis
PCR amplification is achieved by using oligonucleotide primers for flanking regions with a known base sequence.
PCR primers are short pieces of single-stranded DNA, usually around 20 nucleotides in length
Two primers are used per each PCR reaction. They flank the target region to be copied – aka, they are given sequences that cause them to bind to opposite strands of the template DNA.
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Image source: Khan Academy
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Taq polymerase can only make DNA if it’s given a primer – a short sequence of nucleotides that provides a starting point for DNA synthesis
PCR amplification is achieved by using oligonucleotide primers for flanking regions with a known base sequence.
PCR primers are short pieces of single-stranded DNA, usually around 20 nucleotides in length
Two primers are used per each PCR reaction. They flank the target region to be copied – aka, they are given sequences that cause them to bind to opposite strands of the template DNA.
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Major components
Taq polymerase
Primers
Template DNA
Nucleotides
Assemble these components in a tube and put through repeated cycles of heating/cooling to allow for DNA synthesis to occur
Basic steps:
Denaturation: Heating the reaction will separate (denature) the DNA strands to provide the single-stranded template for step 2.
Annealing: Cooling the reaction so primers can bind to their complementary sequences on the single-stranded template DNA.
Extension: Raising the reaction temperature so Taq polymerase can extend the primers, causing the synthesis of new DNA strands.
Repeat this cycle 25-35 times, ~2-4 hours
Can make BILLIONS of copies
Steps of PCR:
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Electrophoresis
We can use gel electrophoresis to see the results of PCR
DNA fragments of the same length form a “band” on the gel that we can actually visually detect with the naked eye
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The Human Genome Project
A 13-year-long project initiated in 1990 to determine the DNA sequence of the entire human genome within 15 years
Met with skepticism early on – would the huge cost of the project outweigh any potential benefits?
Initial goals/principles:
HGP would be an all-inclusive effort to have collaboration from any nation
All human genome sequence info would be freely and publicly available within 24 hours of its assembly
First sequence yeast and worm genomes as a test run for the human genome
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Impact of HGP
HGP has revealed there are ~20,500 human genes
There are 3 billion chemical base pairs
Can help us with:
Genotyping viruses for treatment
Identifying mutations linked to cancer
Medication design
Advances in forensic science
Biofuels and energy
Agriculture advances
The sequence of the DNA is stored in databases available to anyone on the internet
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Measuring Behavior
Dr. Katie Dabrowski, PT, DPT, CSCS
We measure
behavior in
the field and
lab
There are, of course, advantages and disadvantages for both
• We can conduct analyses under “true” environments and conditions
• We can observe behaviors that give us a lot of insight into how a particular group
works
• But we can’t experimentally manipulate
In the field:
• We can control things like temperature, humidity, light/dark cycle, diet, social
environment, age, breeding, genetics, etc.
• But it’s hard to generalized our findings to more populations, given this control
and specific testing populations
In the lab:
Behavioral
Assays in
Mice and Rats
• Monitors spontaneous exploratory behavior when an
animal is placed in an enclosure
• Video-recorded, and therefore allows for measurement of
total activity, average speed, number of activity bouts, etc.
• Example: Mice are recorded in an enclosure with a bright
and dark area, and time spent in each area is measured.
Mice are nocturnal, and choosing an area of bright
illumination over the dark area is a sign of boldness,
whereas the opposite is interpreted as anxiety.
Open field test:
• Animals are trained to use visual cues to swim toward a
hidden platform in a circular pool
• It measures spatial navigation and memory
Morris water maze:
Behavioral
Assays in
Mice and Rats
• Placing a running wheel in an animal’s cage can measure
spontaneous endogenous locomotor activity
Running wheel
• Measures balance by placing rodents on a slowly revolving
rod – literally measure the time it takes for the rodent to
fall off, as a measure of balance
• Can assess the effects of mutations on proprioception and
postural control
Rotating rod
• Assesses an individual’s preference for a particular
substance (usually a drug) by providing an animal a choice
of a container that holds the substance vs. a water control
Preference tests
Controlling experimental variation
To minimize variation, it is essential
to conduct studies such that
measurements are completed at
the same time of day, and under
controlled conditions of
temperature, humidity, air flow, and
illumination
Behaviors of sexes should be
measured separately
Diet and age should be
standardized
Sexual experience of animals
(virgin, pregnant, or sexually-
experienced) can also influence
behaviors
Social environment should be
controlled (animals in isolation
behave differently than those in
crowded conditions)
Genetic background can be a
variable, so in animal studies,
comparing litter mates and inbred
strains can be helpful, or using
twins in human studies
Sources of
variation in
behavior: Genetic
Variation
• We can study genetic variation to identify genes affecting any
treat
• We can create variation via mutagenesis
• Or we can rely on mutations that occurred spontaneously in
nature
• We generally use the term mutation to refer to a lab-generated
allele, and polymorphism to refer to a naturally found change in
allele
How do we determine what genes affect behavior?
Let’s take a look at types of mutant alleles
Types of
mutant alleles
Remember – naturally occurring mutations are called
polymorphisms; mutations are what we call what we do
in labs
• Single nucleotide polymorphisms (SNPs – pronounced snips): Point
mutations that are changes one nucleotide to another
• Mutations can occur in both coding and non-coding regions
• Most common: Substitution of a purine for another purine (A to G,
vice versa) or a pyrimidine for another pyrimidine (C to T, vice
versa)
• Three types of point mutations:
• Silent/synonymous mutations: Do not result in change in the
amino acid, but they may cause changes in the phenotype
• Missense/non-synonymous mutations: Do result in a change in the
amino acid
• Nonsense mutations: Result in premature formation of a stop
codon, therefore generating a truncated protein product – and will
be called a null mutation if the protein is rendered completely
dysfuncional
Types of
mutant alleles
• Frameshift mutation: The pattern of DNA requires the
typical triplet code of nucleotides; this type of mutation
occurs when the total number of nucleotides is not a
multiple of three.
• Large-scale rearrangements in chromosomal structure:
• Gene duplications
• Chromosomal translocations
• Inversions
• Large deletions
Mutation
classification
• Loss of function/null
mutations: When a mutation
abolishes the function of the
affected gene
• Usually recessive
• When the mutation
prevents the survival of
the individual, the
mutation is a lethal
mutation
• Hypomorphic mutations:
Mutations that don’t abolish
the function of the gene
altogether, but reduce the
efficiency of transcription,
mRNA stability, or effectiveness
of the encoded protein’s
function
• Hypermorphic mutations:
Gain-of-function mutations,
which result in a new or
abnormal function of the gene
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