HHMI-NMSU Mobile Molecular Biology Laboratory
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Molecular Biology - Genetics
Popular culture
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Natural Sciences and Agriculture
the Human Genome project. blueprint for a human
• molecular ecology and conservation genetics. snails!
• genetically modified crops and livestock. diet pig. insulin goat. spider goat
•
Medicine
disease discovery and treatment. gene therapy
• genetic counseling. great big Punnet Square
• monitoring viral evolution and transmission. H1N1. HIV. West Nile Virus.
•
Mobile Laboratory Overview
5 day outreach program
2 days discussion (first and final)
• 3 days activities
•
Student Instructions and Supplemental Material (10 min)
Introduction (p 1) and Vocabulary (p S1-2) Molecular
Techniques (p 2)
• Instructions for keeping a Lab Notebook
•
Objectives
• determine phenotype for the “wild type” and eyes absent
mutation in Drosophila
• PCR amplification
• Hardy Weinberg calculations
• gel electrophoresis - to determine genotype
Phenotypes to Genotypes
What is a phenotype?
Phenotype of Drosophila flies that are “wild type” and flies that
carry the eyes absent mutation
Phenotypes are the expression of a genotypes
• DNA codes for genes that express the phenotype
What is DNA?
What is a gene?
Alleles are alternate “versions” (base pair sequences) of a
gene - one inherited from Mom and one from Dad
What is a mutation?
take ~10 min to read the Background for Activity 1 and answer the prelab questions
Phenotypes to Genotypes
Wild type?
Eyeless?
If we know your phenotype for each fly, why bother
looking at the genotype?!
Molecular genetics is a science - science is not SUBJECTIVE
(like an interpretation of a phenotype) it is OBJECTIVE
. . .we look at DNA in order to get an objective determination
of the genotype. . . . AND assess the frequency of mutation
Phenotypes to Genotypes
Wild Type?
Wild Type?
Eyeless?
eya+/eya+
eya+/eya2
eya2/eya2
homozygous
dominant
heterozygous
homozygous
recessive
remember the phenotype is the expression of the genotype
and your genotype is defined by your two alleles for the eyes
absent gene (eya+ is normal eya2 is mutated)
?? What HYPOTHESIS can you make about each genotype,
now knowing the phenotypes ??
What the genes may mean. . . . .
Drosophila flies are a “model organism” used to understand
the effects of mutation on form and function (humans have a
gene similar to the eya2 on chromosome 20)
Mutations (deleterious alleles) can be present in a population
but undetected because they occur in a heterozygous
genotype (but still can be passed to offspring)
Understanding genes responsible for disorder and disease is
important for medical applications - change over time in
frequency of the alleles is the evolution of a disease (or a virus
like HIV or H1N1)
Phenotypes to Genotypes
DNA extraction
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smash
identify
spin
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3-3
Q uickTim e™ and a
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3-2
3-1
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boil
spin
Phenotypes to Genotypes
PCR amplification - of the whole fly genome?
5´
3´
3´ step 1. Samples are heated
(95°C) to denature the double
stranded DNA so the nucleotides
5´ of each strand are exposed.
5´
3´
3´
step 2. Samples are cooled
slightly (68°C) so the primers
can anneal to the single stranded
5´ DNA.
5´
3´
step 3. Samples are heated
slightly (72°C) so that the PCR
enzyme (polymerase) can extend
the strand from the primers
3´
5´
The next step in DNA analysis
beyond fragment analysis
DNA is used to determine changes in allele frequency over
time in a population (evolution)
Hardy-Weinberg Equilibrium (Activity 4)
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decompressor
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QuickTime™ and a
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phenotypes and genotypes to determine if a population is evolving
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decompressor
are needed to see this picture.
p+q=1
p2 + 2pq +q2 = 1
Phenotypes to Genotypes
Gel electrophoresis
sample (each PCR) is loaded into a
gel matrix (agarose)
• electric current is applied and
fragments are separated by size
• visualized under UV light
•
Electrophoresis Chamber
power supply
large fragments
+
small fragments
Tomorrow
take a few minutes to read over pages 9-13
(1) extracting DNA, (2) setting up a PCR
kit contents - at each lab bench
Using a micropipetter
take a few minutes to read over page 6
parts
and function
how to
hold it
how NOT
to hold it
proper
operation
See you tomorrow
Keeping a Lab Notebook
Recording procedures, notes, and observations as the work
is being done. *Facebook Status or Twitter Tweet
3 main parts to a lab notebook entry: Purpose Procedure
(Methods) Outcome (Results)
Purpose: 1-2 sentences what you’re about to do
Pur po s e: Extra c ti ng DNA fr om Dro s ophila f lies f o r
later u se i n a PCR.
Proc edure :
Procedure: what you’re doing as you’re
doing it and observations (not a set of
instructions)
Grou p #: 3
labeling tu bes and r ec or ding pheno ty pe:
tu be
phen o t ype
1-1
a.
1-2
a.
1-3
a.
1-4
a.
1-5
a.
wi ld t ype ( red)
wi ld t ype ( red)
e yeles s
wi ld t ype ( red)
e yeles s
putti ng f ly int o tu be
Outcome: observed results, may include visuals
Res ult : DNA extr act ed f ro m c ells and pres ent i n the
u pper l ayer o f th e bu f fer so lut io n.
DNA (deoxyribonucleic acid) is an
information molecule that provides
the instructions for making proteins
• double stranded, double helix
•
made of nucleotides
each set of nucleotides is
called a base pair (bp)
•
fragment size from counting
base pairs
•
DNA fragment sizes may vary
if there is an insertion or
deletion
•
http://www.scq.ubc.ca/a-monks-flourishing-garden-the-basics-ofmolecular-biology-explained/
A gene is a “unit of inheritance”
located on a specific
chromosome (the eyes absent
gene is on a small region of
chromosome 2 in Drosophila)
• each diploid organism has
two copies of a gene - one
inherited from their father and
one from their mother
• eyes absent gene plays a
role in eye formation mutation in this gene leads to
Drosophila flies with no eyes
on head
•
http://en.wikipedia.org/wiki/Gene
Mutation “a change in DNA sequence” - may cause
a change in a gene . . affecting the amino acid
sequence . . . affecting the protein produced. . .
effecting the phenotype
could be a change in one nitrogenous base to another
A
T or G
C
could be an insertion of a portion of DNA or loss of a portion
of DNA (deletion)
eyeless mutation in Drosophila caused by a deletion