this is all of the information that I have please help Lab 5 Introduction - Genetic mapping See figure 5.1 for a schematic of the fly the cross you initially started with, you'll either crosses you have been working on. Two labs ago, map the distance between the w gene and the m you set up a pair of reciprocal parental crosses, gene, or between the w gene and the y gene. between mutant and wild type flies (fig. 5.1a). You had one of two different mutant strains, each with two mutant phenotypes - either white eyes all F2 individuals will receive only recessive (w) and miniature wings (m), or white eyes (w) alleles from this parent (fig. 5.1d, orange and and yellow body (y). The phenotypes of the F1 yellow chromosomes). Because of this, the flies should have indicated to you that all mutant phenotype of each F2 fly will tell you which phenotypes in question are x- linked recessive alleles (mutant or WT) were inherited from the (fig. 5.1b). heterozygous female F1 parent (fig. 5.1d, dark and light blue chromosomes). The first F2 fly Last lab, you used the F1 flies from one of shown in figure 5.1d inherited 'a B' from the your parental crosses to set up an F1 cross (fig. heterozygous parent and will end up with the consequence. After crossing two pure breeding this F2, you observe a ABphenotype and parents, F1 offspring will be heterozygous for therefore know that this fly received 'A B' from nearly all genes in question - the exception is X - the heterozygous parent. linked genes in the male offspring. Since the Y chromosome is equivalent to recessive alleles for The goal of genetic mapping is to X-linked genes, these F1 males are recessive for determine the likelihood of cross over between all X-linked genes and act as a test cross. The two loci/genes. If we score the phenotypes of a heterozygous F1 females and recessive F1 males large F2 population from our crosses, we can (test cross) can be used to map the distance determine the recombination frequency of your between the genes causing the two phenotypes two genes. of your parental mutant female. Depending on e) F2 phenotype scoring: f) Recombination frequency: Eigure 5.1: Schematic of your Drosophila crosses, See text of lab 5 intro for description. For a given F1 gamete for the F2 individual it number of flies, it is easy to calculate the creates), if no cross over occurs between the two recombinant frequency between your two genes genes in question, the F2 phenotype will be the (fig. 5.1ef ). same as one of the original parents - either fully WT or double mutant in this case. In figure 5.1d Recall from last time that a lower recombinant these have blue chromosomes of a single colour. frequency is observed when genetic map If a crossover does occur between the two genes, distances are small. When genes are close to the F2 fly will have a phenotype unlike either of each other, there's a narrow range on the the parents - a recombinant phenotype (shown chromosome for a random crossover to land.

1. this is all of the information that I have please help Lab 5 Introduction - Genetic mapping See
figure 5.1 for a schematic of the fly the cross you initially started with, you'll either crosses you
have been working on. Two labs ago, map the distance between the w gene and the m you set up
a pair of reciprocal parental crosses, gene, or between the w gene and the y gene. between mutant
and wild type flies (fig. 5.1a). You had one of two different mutant strains, each with two mutant
phenotypes - either white eyes all F2 individuals will receive only recessive (w) and miniature
wings (m), or white eyes (w) alleles from this parent (fig. 5.1d, orange and and yellow body (y).
The phenotypes of the F1 yellow chromosomes). Because of this, the flies should have indicated
to you that all mutant phenotype of each F2 fly will tell you which phenotypes in question are x-
linked recessive alleles (mutant or WT) were inherited from the (fig. 5.1b). heterozygous female
F1 parent (fig. 5.1d, dark and light blue chromosomes). The first F2 fly Last lab, you used the F1
flies from one of shown in figure 5.1d inherited 'a B' from the your parental crosses to set up an
F1 cross (fig. heterozygous parent and will end up with the consequence. After crossing two pure
breeding this F2, you observe a ABphenotype and parents, F1 offspring will be heterozygous for
therefore know that this fly received 'A B' from nearly all genes in question - the exception is X -
the heterozygous parent. linked genes in the male offspring. Since the Y chromosome is
equivalent to recessive alleles for The goal of genetic mapping is to X-linked genes, these F1
males are recessive for determine the likelihood of cross over between all X-linked genes and act
as a test cross. The two loci/genes. If we score the phenotypes of a heterozygous F1 females and
recessive F1 males large F2 population from our crosses, we can (test cross) can be used to map
the distance determine the recombination frequency of your between the genes causing the two
phenotypes two genes. of your parental mutant female. Depending on
e) F2 phenotype scoring: f) Recombination frequency: Eigure 5.1: Schematic of your Drosophila
crosses, See text of lab 5 intro for description.
For a given F1 gamete for the F2 individual it number of flies, it is easy to calculate the creates),
if no cross over occurs between the two recombinant frequency between your two genes genes in
question, the F2 phenotype will be the (fig. 5.1ef ). same as one of the original parents - either
fully WT or double mutant in this case. In figure 5.1d Recall from last time that a lower
recombinant these have blue chromosomes of a single colour. frequency is observed when
genetic map If a crossover does occur between the two genes, distances are small. When genes
are close to the F2 fly will have a phenotype unlike either of each other, there's a narrow range

2. on the the parents - a recombinant phenotype (shown chromosome for a random crossover to
land in to in the figure as having dark and light blue lead to a recombinant offspring. As a class,
you'll chromosomes). In your case, the recombinant score the F2 populations from the w - m
cross flies will have one mutant phenotype but not and the wy cross. Youll calculate the both (eg.
white cyes but normal wings/body recombination frequency for each, which will let colour).
After scoring the phenotypes of a large you compare these two map distances. Lab 6 Introduction
- Restriction fragment length polymorphism The gene you will be analyzing in this lab is
individuals to metabolize caffeine quickly and called CYPIA2. The "CYP portion of this gene's
others to metabolize it slowly. In slow name refers to the fact that it encodes a metabolizers,
caffeine remains in the body cytochrome P450 protein. Cytochrome P450's longer, and therefore
has a greater physiological make up a very large enzyme family, with impact than it would to a
fast metabolizer. Can representatives being found across all domains you guess whether you are
a fast or a slow of life. The various P450 enzymes have highly metabolizer of caffeine? varied
substrates and work in an enormous number of different biological roles. One well As mentioned
above, the two alleles you will known role of these enzymes is to metabolize be assessing differ
by only a single base. This drugs and other xenobiotics (foreign type of allele is called a single
nucleotide compounds that are not created in the body). polymorphism, or SNP (pronounced
"snip"). Like many xenobiotic metabolizing enzymes, between individuals. An estimated 10
million CYP1A2 acts on a variety of substrates. Some of SNP's exist in the human genome, or
this protein's well-known substrates are approximately one SNP in every 300b. SNP caffeine,
acetaminophen (Tylenol), and aflatoxin analysis is potentially useful for many different B1.
CYP1A2 is expressed in the liver, and its goal purposes, including predicting diseases is to
modify its substrates into more hydrophilic susceptibly, or use as molecular markers in
compounds, making them easier to excrete by agricultural breeding programs. the kidneys. The
phenotype associated with these alleles Previous research has indicated that the is not easy to
score, so you will determine your CYP1A2 gene has multiple alleles in the human genotype with
PCR and agarose gel population. The two alleles you will examine electrophoresis, like you did
in lab 2. The today differ by only a single base pair. At a given difference between alleles this
time, however, is position, one allele has an adenine nucleotide much more subtle than those
analyzed last time. and the other allele has a cytosine. These two The small variation between
alleles will not alleles have been associated with differing rates affect the size of the PCR
fragment, so you of caffeine metabolism. This causes certain will need to use another technique,
called BIOL 2014 Lab 5 - Gene mapping, and Lab 6 - RFLP 3
restriction fragment length polymorphism a given DNA fragment, as well as the location of
(RFLP) to discern the two alleles. the cut site. Apal and other restriction enzymes RFLP analysis

3. differentiates DNA sequences are also useful in other molecular biology that affect the
recognition sequence of techniques, namely recombinant DNA restriction endonucleases (often
casually called technology, which you will learn about it the 'restriction enzymes'). Restriction
endo- lecture portion of this course. For these other nucleases are proteins that originated as a
techniques, additional features of restriction defense strategy for bacteria, but have been enzymes
are important, though they will not be exploited for various purposes in molecular discussed
here. biology. The term endonuclease implies that RFLP analysis is able to distinguish SNP these
enzymes cut DNA within a strand (as alleles when the differing base occurs within the opposed
to exonucleases, which remove bases restriction enzyme's recognition sequence. In from the end
of a DNA strand). Bacteria use one allele, the recognition sequence will be these enzymes to
protect themselves from intact, and the enzyme will cut the DNA viruses by cutting up the
invading viral DNA. fragment into two smaller pieces. In the other Molecular biologist,
however, use these allele, the recognition sequence will be altered, enzymes for their ability to
modify DNA meaning that the enzyme will not cut and will molecules in a precise and
predictable manner. leave one large fragment. This concept, as well The key feature that makes
restriction at the specific fragments sizes you should enzymes useful in molecular biology is that
they observe in today's experiment are shown in utilize specific recognition sequences. A figure
6.1. Test your understanding by drawing restriction enzyme will only cut a DNA strand in the
expected banding patterns for each of the that contains its recognition site. The three possible
genotypes on the right of the enzyme you will use today is called Apal and figure. will cut DNA
only if it has the specific Last lab you used provided genomic DNA as a recognition sequence 5'-
GGGCCC-3'. For PCR template to amplify a region of the CYP1A2 their use in RFLP analysis,
the most important gene. Today, you will perform RFLP and agarose characteristic of a
restriction enzyme is that it is gel electrophoresis analyses to determine the predictable in terms
of whether or not it will cut genotypes of your provided DNA.
1-Chromosome 2-PCR amplifies a small repion centered 3-Apal cuts fragments with the C allele
15 in your around aNP. One allele has the Apol into 275 and 187bp pieces. Fragments genomic
DNA recognition sequence & the other with the A allele are left as a single prep. doesn't 462b
piece. B) Eigure 6.1: Overview of the DNA analysis that will be used in today's experiment. The
expected fragment sizes are given in (a-3). Test your understanding by drawing the banding
patterns you would expect to observe on your agarose gel in (b). On the left is the DNA ladder
with known band sizes indicated (in bp). Three empty lanes are shown, representing the three
possible genotypes that an individual could have. Protocols Analyzing F2 fly populations (Lab 5)
1. Your group will be provided an F2 population from your previous F1 cross. If the flies are not
already anaesthetized, use FlyNap to knock them out, and pour into one or more petri dishes. DO

4. NOT leave the FlyNap in your vial for more than 3 minutes. 2. Score the phenotypes of the flies
your group received using the dissection microscope. Depending on the cross you are working
with, you will either be scoring for eye colour & body colour, or eye colour & wing length.
You do not need to differentiate males from females for this analysis. 3. Compile your group's
data and provide it to a TA. Using the compiled class data, calculate the recombination
frequency for each cross. BIOL 2014 Lab 5 - Gene mapping, and Lab 6 - RFL.P 5
Purify DNA with a spin column (Lab 6) You will use a spin columa kit to purify the DNA from
your completed PCR reaction. A spin column contains a filter that will bind DNA under certain
conditions, and release the DNA under other conditions. Liquid is put into the spin column, then
it's centrifuged to pass the liquid through the filter. Flow chart Detailed protocol Mix DNA
binding buffer into your PCR, then pipette 1. Find the PCR reaction than you set up and ran the
mixture into a spin column last lab on the side bench or your table. - This buffer contains salts
that cause the DNA to stick to the spin column's filter 2. Transfer your 50pl PCR reaction into a
micro tube and mix in 300l of DNA binding buffer. - The spin column should be sitting inside
the collection tube Transfer the full 350 l volume into a spin column 3. Spin your column in a
microcentrifuge for Centrifuge to pass the buffer through the filter 30sec1min. - The DNA sticks
to the filter, while the buffer and all the leftover PCR ingredients pass into the 4. Remove the
spin column dump the flow coliection tube to be discanded through into the liquid waste beaker,
and replace the spia column iato the collection tube. 5. Add 660l of DNA wash buffer to your
column. Pipette DNA wash buffer iato the spin columa, then and spin again for 30 sec-1min. Put
the lid back centrifuge again into the collection tube on the bottle of wash buffer quidily- don't -
The DNA stoys in the filter, while any remaining leave it open longer than necessary. sults are
washed away 6. Discard the flow through, return the spin - Again, discard the liuid column to the
collection tabe, and spin for another 3min. Add distilled water to the spin column, place it in a 7.
Transfer your spin column to a new labelled microtube, then centrifuge again microtube. Add 40
pl dH 0 directly on to the - Distilled water reicases the DNA from the filter filter at the bottom on
the spin column - After spinning you will have purified DNA in the (make sure it doesn't stick to
the side]. microtube 8. Spin your column inside the microtube (not the collection tube) for 30
sec-1min to elute your purified DNA The lid of the microtube will be left open for this spin. 9.
Your purified DNA is in now in the liquid in your microtube. Discard the spin column and
collection tube.
Question 7 (2 points) The Apal enzyme is supplied at a concentration of 10 units/ I. To prepare
for the lab, 50l of enzyme were diluted with 350IdH2O. It's this diluted enzyme you used in
today's protocol. How many units of Apal enzyme did you add to your reaction? (enter numbers

5. only!) A units Question 8 (1 point) What total x2 did you calculate for your X2 test? Enter just a
number rounded to 2 decimal places. A
You perform today's RFLP experiment using 5 different control reactions, and get the results
shown here. Assuming that everything was set up correctly, indicate which control is loaded in
each lane. No DNA control 1. Ladder 2. Ctr1 DNA with genotype AC 3. Ctrl2 Should not be
possible 4. Ctrl3 DNA with genotype AA 5. Ctrl 4
How would the above gel look if you made the following mistakes (assume that only 1 mistake
is made in each case). (Brightspace may change the order of options from printed Worksheet)
Two of your answers from the last question should be the same. Suggest a way that you could
differentiate between these two cases. Select all answers that apply Select 2 correct answer(s)
You could run the PCR on a gel directly, without first using a spin column You could try running
thegel for longer to get better separation of the bands You could sequence the DNA encoding
your Taq polymerase enzyme You could use a spin column to try to purify another DNA sample
(e.g. some plasmid DNA you had leftover from lab 1) You can try the experiment again and
double check your pipette setting and technique Question 6 (1 point) What is the genotype at this
SNP for the genomic DNA you were provided? AA AC CC