Practical:

Drosophila care
Black - males white-females
NCBI databases. Be able to find basic gene-related information on NCBI.
Genomic region - gene structure (box = exon , lines = intron)

Box, light green = untranslated, dark green protein coding region

Perform and analyze a BLAST search

Pipetting accuracy

Concepts and methods

Explain PCR steps, the reason for primers

Polymerase chain reaction (PCR) - makes lots of copies of seg of DNA
PCR is really an in vitro DNA replication reaction. A short segment of DNA is selectively
replicated million of times. This technique allows researchers to detect, analyze, and measure a
defined region of DNA within a complex mixture

Primers- small anchors known on both sides of the region to be amplified. Primers are short
single strand DNA pieces with a sequence complementary to the edges of the target region.

There are three steps necessary for the DNA to be replicated:

1) open the double-stranded DNA to make it single stranded = denaturation
2) attach the primers to the specific complementary sequence = annealing
3) copy the DNA strand starting at the primer in a 5’ to 3’ direction = extension (elongation)

PCR STEPS Repeat for 30 times

These three steps can be accomplished by changing the temperature of the reaction mixture
necessary for
1) denaturation at 94-95oC
2) annealing between 50 and 65oC
3) extension 72oCThis “cycling” is easily done by use of a special machine known as a
thermocycler.
Explain principle of gel electrophoresis
 Gel electrophoresis allows separation of macromolecules from a mixture based on a physical
property of the molecules (charge, shape, size, etc.). The separation is done when the
macromolecules travel within a solid matrix (gel) using electricity. DNA, RNA and protein
molecules can be separated by gel electrophoresis.

The mixture samples are loaded into the wells of the gel, which is placed into an
electrophoresis chamber filled with buffer solution.

A buffer solution both within and around the gel conducts the electricity. Because DNA is
negatively charged, it will be pulled towards the positive electrode.
The DNA mix is loaded on “top” of the gel in “wells”, and then the fragments are pulled inside
the gel by the electrical current.

different DNA fragments can be separated based on their mobility, which is a reverse
correlation with their size size or their topology

The lower the % of agarose used, the bigger the open spaces, and the larger the DNA
molecules that can travel through the gel. Typical % that we will use varies from 0.8% (optimal
from 300 base pairs (bp) to 1 kilobase (kb) to 1.2 % (optimal from 100 bp to 6 kb).
After separation, the DNA fragments can be:
1) stained directly inside the gel using a dye for DNA molecule. We will use a chemical known
as GelRed. After staining, the DNA appears as “bands” in the gel.
2) processed for further techniques such as Southern blotting (for hybridization).
3) Excised from the gel for cloning, sequencing, etc.

Understand gene structure and parts.

Discuss different types of polymorphisms and analyze gel data for SSR and SNP
polymorphisms
Polymorphisms and simple mutations are random events. So often they are NOT located within
genes, and do not automatically have a visible effect on the phenotype

Simple polym:
SSLP: Simple Sequence Length Polymorphism in microsatellite regions (AKA VNTR: Variable
Number of Tandem Repeats)
•SNP (say “snip”): Single Nucleotide Polymorphism

•RFLP (a subtype of SNP): Restriction Fragment Length Polymorphism

The sequence for a specific Restriction Enzyme (RE) site is recognized by the enzyme and the
DNA is cut.
After gel electrophoresis, two fragments result from this region of the genome.

A minor difference in the DNA sequence of two molecules can be detected if the difference
eliminates (or creates) a restriction enzyme site

After restriction digest, the fragment mixture is separated by gel electrophoresis. The two
chromosomes of a heterozygous can be distinguished.
In this example HaeIII is the restriction enzyme. Because of SNP, one site is present in Bob, but
not Joe.

RFLPs are used in paternity tests, forensic analysis, genetic testing, etc..
•CNV , Indel, and segmental duplications

Most common
Simple Sequence Repeats (SSR)

Many genomes contain regions of DNA where the sequence is made of

short units repeated many times in tandem (usually 5-50 times). For example:
CACACACACACACACACACA. These regions are known as simple sequence repeats (SSR)
or microsatellites. They are found at many positions on the chromosomes.

The number of repeats found at a given chromosome position varies between individuals or
even within one person between homologous chromosomes (heterozygous). The associated
polymorphism is known as Simple Sequence Length Polymorphism (SSLP, aka VNTR: Variable
Number of Tandem Repeats).
6 repeats: AGAAGAAGAAGAAGAAGA
9 repeats: AGAAGAAGAAGAAGAAGAAGAAGAAGA

The important point to remember is that the DNA sequence on either sides of the SSR have a
unique sequence specific for that position on the chromosome. So primers for PCR and/or
known restriction enzyme sites can be used to determine the size of the entire SSR region
following gel electrophoresis.

The pattern (size and number) of bands following gel electrophoresis indicates whether the
organism is homozygous for a certain number of repeats or heterozygous

SSR/microsatellite can be found both within or outside of genes.

 (CNV or CNP)

PCR and DNA sequencing

Microarray

Small simple changes in the DNA sequence that can only be detected using molecular
techniques.
The DNA sequence at a particular position within the genome can be different between people
or even between the 2 homologous chromosomes of one person.

More than 5 million common SNPs each with frequency 10-50% account for the bulk of human
DNA sequence differences. The majority of human sequence variations are due to substitutions
that have occurred ONCE in the history of mankind at individual base pairs, SNPs (Patil et al.
2001). There is about 1 SNP in every 600 base pairs. It is estimated that ~60,000 SNPs occur
within exons; 85% of exons are within 5 kb of nearest SNP.
The more critical points to understand are:
1.at the molecular level polymorphisms and simple mutations (substitution, indel) are the same.
2.Polymorphisms and simple mutations can only be distinguished based on their frequency in
the context of a population.

SNP
Single Nucleotide Polymorphism
Imagine a gene (blue and red bars on the chromosomes), and the following sequences in the
promoter region of the gene: (differences are underlined):
ACCAATGGACTAG (green chromatids)
ACCGATGGCCTAG (orange chromatids)
These 2 sequences would be allelic variations of that gene.
Single Nucleotide Polymorphism

Comparing the sequence from different organisms allows the detection of polymorphisms, in
this example, a C/T polymorphism

Hybridization can be performed such that 100% match is required for the target-probe hybrid to
form. One base difference would prevent hybridization.

Be able to discuss different types of mutations and their effects on the transcript and
protein
Spontaneous mutations
•By unknown exposure to physical or chemical agents
•By uncorrected errors during the process of DNA replication/repair (image below).
•By movement of transposable elements
•Probably other unknown possibilities

Somatic versus germline

•A somatic mutation affects only certain cells of an entire individual and is NOT passed on to
the next generation. These kinds of mutations are often responsible for some types of cancer or
other diseases.
•A germ line mutation occurs within the DNA of a gamete and can be passed on to the
progeny.
These changes in DNA sequence do occur at random places in the genome. In many species
genes only occupy a small percentage of the genome (2% in humans), and most mutations
occur outside of genes.

Base substitution
Many of these are the basis for Single Nucleotide Polymorphisms (SNP, say “snip”).
Their effects can be from none (the majority of them) to extremely deleterious (sickle cell
anemia, etc..)

Base additions (insertions) or deletions: a few to many nucleotides are removed or added at
one point in the sequence. There are many different causes or mechanisms for these mutations
to happen.

Expanding trinucleotide repeats are sequences where 3 bases are repeated next to each other
for a variable number of times. Over-repetition can be the basis for several genetic

oBecause mutations / polymorphisms occur randomly, they can change sequences both inside
(intragenic) or outside of genes (intergenic).
oBecause of the often low percentage of genome DNA dedicated to genes, most mutations/
polymorphisms are probably intergenic.

Effects of intragenic mutations

How do mutations affect the production of the right amount of a functional final product, either
proteins (for coding genes) or RNA (for non-coding genes).
Mutations in the RNA coding region (from TSS to transcription stop), will typically NOT affect the
actual transcription of the gene and its regulation, i.e. the RNA will still be produced. But what
about the protein??

After RNA is produced and processed, it will arrive at the ribosomes and translation will start. If
the change in the DNA sequence does not change the encoded amino acid (because of genetic
code wobble), there is no effect at the protein level and this is a silent (synonymous) mutation.

If the mutation changes to a codon for a different amino acid, the mutation is a missense
mutation. The protein is still translated, but has a different amino acid. We will consider possible
phenotypic effects later.

If the mutation creates a stop codon, the mutation is a nonsense mutation. The translation
stops and the rest of the protein is not made. This produces a shorter protein. We will consider
possible phenotypic effects later.

The effect of a missense mutation on a protein is very difficult to predict, but some evaluation
can be done based on:
1) How different is the missense amino acid from the original one.
2) Where the change is in relation to the protein functional domains and tertiary/quaternary
structure.
3) Whether the change is affecting an amino acid which is modified post-translationally.
Missense mutation and protein function

1) How different is the missense amino acid from the original: this can be assessed by checking
whether the “old” and “new” amino acids belong or not to the same amino acid group. So for
example a Asp to Glu mutation does not change the group and is known as a “conservative”
mutation.

However a Lys to Asp change is a non-conservative missense because it changes the amino
acid group involved. A lysine can work as a H donor and forms a Hydrogen or a ionic bond with
a negatively charged amino acid in the 3ary structure. Replacing Lys with Asp, which is
negatively charged would prevent that 3ary structure bond and may have an effect on the
protein shape-function.

If the insertion/deletion is a multiple of 3, then the reading frame for the protein is not changed
(in-frame) and the effect is the addition or loss of 1, a few, or many amino acids.
•If the insertion is other than a multiple of 3, then there is a change in the reading frame and
the mutation is called a frameshift mutation. Typically no functional protein will be produced or
the protein will be very unstable.
•Insertion/deletion are often referred to as “indel”

If the number of bases added or removed is a multiple of 3, the way the sequence is read by the
ribosome will not changed, but one to a few amino acids will be added/removed.
This can still be harmful to the protein shape-function.

If the number of bases added or removed is not a multiple of 3, and because the ribosome
reads the mRNA in codons of 3 bases, the way the sequence is read by the ribosome will be
shifted, AKA frameshift mutation.
Explain how different alleles of one gene can lead to different phenotypes (e.g. white
alleles) depending on the effect of the mutation on the protein.

Explain how mutations in different genes can have the same phenotype and the principle
of complementation

In genetics, complementation occurs when two strains of an organism with different homozygous
recessive mutations that produce the same mutant phenotype (for example, a change in wing
structure in flies) produce offspring with the wild-type phenotype when mated or crossed.
Complementation will occur only if the mutations are in different genes. In this case, each strain's
genome supplies the wild-type allele to "complement" the mutated allele of the other strain's
genome. Since the mutations are recessive, the offspring will display the wild-type phenotype. A
complementation test (sometimes called a "cis-trans" test) can be used to test whether the mutations
in two strains are in different genes. Complementation will not occur if the mutations are in the same
gene. The convenience and essence of this test is that the mutations that produce a phenotype can
be assigned to different genes without the exact knowledge of what the gene product is doing on a
molecular level.

A complementation test brings two mutant genes together in the same cell or organism.
This example uses genes involved in a pathway for pigment formation. When either enzyme is non
functional, no pigment is made.

In a cross between the two mutants, if the progeny is mutant (NO COMPLEMENTATION), then it
means that there is no good copy of the gene: the two mutations are alleles of 1 gene. In a cross
between the two mutants, if the progeny is wild type (COMPLEMENTATION), then it means that
there is one good copy of each genes: the two mutations are in separate genes, there are NOT
alleles.
-On the molecular level, complementation typically occurs between
epistatic alleles.
-Epistasis describes the relationship between two genes that are
dependent on each other in achieving a certain phenotype
-These genes usual code for proteins use at different point in the same
metabolic pathway

Distinguish penetrance from expressivity.

Penetrance refers to the probability of a gene or trait being expressed. In some cases, despite
the presence of a dominant allele, a phenotype may not be present. One example of this is
polydactyly in humans (extra fingers and/or toes). A dominant allele produces polydactyly in
humans but not all humans with the allele display the extra digits. “Complete” penetrance
means the gene or genes for a trait are expressed in all the population who have the genes.
“Incomplete” or ‘reduced’ penetrance means the genetic trait is expressed in only part of the
population. The penetrance of expression may also change in different age groups of a
population. Reduced penetrance probably results from a combination of genetic, environmental,
and lifestyle factors, many of which are unknown. This phenomenon can make it challenging for
genetics professionals to interpret a person’s family medical history and predict the risk of
passing a genetic condition to future generations.

Expressivity on the other hand refers to variation in phenotypic expression when an allele is
penetrant. Back to the polydactyly example, an extra digit may occur on one or more
appendages. The digit can be full size or just a stub. Hence, this allele has reduced penetrance
as well as variable expressivity. Variable expressivity refers to the range of signs and symptoms
that can occur in different people with the same genetic condition. As with reduced penetrance,
variable expressivity is probably caused by a combination of genetic, environmental, and
lifestyle factors, most of which have not been identified. If a genetic condition has highly variable
signs and symptoms, it may be challenging to diagnose.