Department of Biological and
Environmental Sciences
BIO L320 – GENETICS LAB
Dr. Jacqueline Jones
Laboratory 5 – Mendelian Genetics – Principle of Independent Assortment
lab and Principles of DNA Sequencing
Principle of Independent Assortment: In this section of today’s lab, we will conduct two
exercises related to Mendel’s Principle of Independent Assortment (Investigation 1):
Exercise 1. The first experiment is a pen and paper exercise based on the dihybrid cross for
Drosophila ebony body X vestigial wing described on page 11 (old book) & page 13 (new
book), part A, number 1. You need to calculate the expected results for the F1 phenotypes,
and F2 phenotypes and frequencies as described on page 11 (old book) & page 15 (new book),
part A, number 2. In addition, you need to show (a) the genotype of the F1 flies resulting from
this cross, (b) the haploid gametes that the F1 individuals would produce, and (c) produce a
Punnett square for the F2 generation of this dihybrid cross, showing all genotypes and
phenotypes. You will have to write this part up on a sheet of paper, DO NOT use the lines at
the bottom of page 11 (old book) & page 15 (new book). (25 points)
Principles of DNA Sequencing:
Exercise 2. The purpose of this exercise is to provide detailed instruction on the practice of DNA
sequencing. You will be provided an actual autoradiograph (an exposed sheet of x-ray film) of a
DNA sequencing gel for analysis. The sequence deduced from the autoradiogram will differ
from a wild type sequence by a single nucleotide(s). Refer to the handout provided on
Blackboard. This difference represents an actual mutation in the DNA molecule. You are
expected to identify the location(s) of the mutated nucleotide(s). You can also work in groups to
read the sequence and compare results to assure accuracy. Make sure you read the handout
because you will see this information again on your midterm. (25 points)
Part 1 does not require a formal lab report. Just give your answers and the results of the cross. For part 2, a type
written lab report is needed. Use the handout to help you put together your report. You MUST have a cover page.
Total = 50 points
Pages needed to be copied from the lab book: 11 and 13
(old book); 13 and 15 (new book). Also Print out other
Document for Exercise 2.
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Principles of DNA Sequencing Part II
Background Information
The purpose of this exercise is to provide detailed instruction on the practice of DNA
sequencing. The student is provided an actual autoradiograph (an exposed sheet of x-ray
film) of a DNA sequencing gel for analysis. The sequence deduced from the autoradio
gram will differ from a wild type sequence by a single nucleotide. This difference rep
resents an actual mutation in the DNA molecule. The students are expected to identify
the location of the mutated nucleotide. Students should also work in groups to read the
sequence and compare results to assure accuracy.
Rapid analysis of DNA sequence was developed during the 1970's from research groups in
the United States and England. Since its early days, these methods have been refined and
automated.
There are two basic approaches to DNA sequence analysis. One involves a set of organic chemical reactions with the DNA bases. The other uses an enzymatic process. The
chemical method is tedious and labor-intensive, whereas the enzymatic approach, which
is often called the dideoxy method, is quite fast. The autoradiographs you have been
given as part of this experiment, are the result of the enzymatic procedure which uses the
Kienow fragment of the E.coli DNA polymerase I to make a DNA copy of the region to
be sequenced. More commonly, a DNA polymerase known as sequenase or equivalent is
used for DNA sequencing.
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A specialized cloning vehicle constructed from an E. co/ivirus, called M13, facilitates rapid
DNA sequence analysis. This virus contains a polylinker, which is a short region of DNA,
about 57 base pairs, containing several unique restriction sites. Segments of DNA to be
sequenced are inserted into the polylinker region using standard cloning procedures (Figure 1}.
The M13 virus contains a single-stranded circular genome
with about 7200 nucleotides. The virus will infect E. coli strain
JM101 cells which contain a fertility factor. These cells are F·,
and male. The virus infects by attachment to the sex pilus.
Shortly after infection, the viral DNA will become double
stranded. It is this form which serves as a template for produc
tion of single-stranded DNA progeny. The DNA associates with
the viral proteins to form mature virus and the virus exits the
cell by budding; the cell is not lysed. For cloning and sequenc
ing purposes, double-stranded DNA to be sequenced is inserted
into the M13 polylinker region of the DNA intermediate, and
then transformed into competent JM101 E.coli cells. The trans
formed cells will begin to produce progeny virus.
polylinker region
.--::w..�----
To sequence DNA which has been inserted into the polylinker region of M13 single
stranded DNA is prepared from viral plaques. In this experiment, a short 17-base synthet
ic single-stranded DNA is allowed to hybridize (form a base pairing} with a unique site in
M13 adjacent to the polylinker.
I
II
site of hybridization
of sequencing
primer
Figure 1
Experiment Overview and Procedures
EXPERIMENT OBJECTIVE:
The objective of this experiment is to develop an understanding of DNA sequencing and
analysis. This is a dry lab which contains autoradiographs from an actual DNA sequencing
experiment.
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EXPERIMENTAL PROCEDURE:
1.
Obtain the sample autoradiograph and place it on a light box to enhance
visualization.
2.
The sequencing reactions have all been loaded in order: G-A-T-C.
3.
Begin analysis of the DNA sequence at the bottom of the autoradiograph with the
circled band, which is an A.
4.
Identify the location of the mutant nucleotides. What was the mutation? Is there
more than one mutation?
5.
To help you start, the sequence begin with an 5' A.
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Drosophila and Maize Experiments in Genetics: Monohybrid and Dihybrid Crosses
11
1. What F2 phenotypic ratio do you expect to obtain?
2. What F2 genotypic ratio is expected?
3. Which trait is dominant in your experiment?
4. How do you know?
B. Maize Crosses
The inheritance of the extensive genetic variability in maize also obeys Mendelian principles; both
seedling and aleurone (endosperm) characters lend themselves to classroom use. Figure 1.7 shows a
flat of F2 seedlings segregating for a recessive mutant allele for albinism (absence of chlorophyll). The
classical Mendelian ratio of 3:1 is expected. Similarly, Figures 1.8 and 1.9 show ears of F2 kernels of
corn that are segregating in a ratio of 3colored : 1colorless aleurone typical of a monohybrid cross, or
in a ratio of 1colored : 1colorless aleurone typical of a monohybrid testcross. Can you use the photos
to confirm these expectations?
Because maize has a relatively long life cycle (3 months or more to complete), you probably will
not be able to conduct actual experimental matings with it. Your instructor might provide you with ears
of genetic corn or flats of F2 seedlings and request that you determine the number of individual seeds or
plants having the various phenotypes and then interpret the data in terms of Mendelian principles. For
example, suppose you counted 40 green and 12 albino seedlings in an F2 population. How would you
interpret these data?
FIGURE 1.7 Flat of F2 corn seedlings segregating for a recessive albino mutation.
The classical Mendelian ratio of 3:1 is expected.
Drosophila and Maine Experiments in Genetics Monohybrid and Dlhybrid Crosses 13
TABLE 1.3 Record of F, Data for Maize Indosperm Trait
Number
of Kernels
Observed
Phenotypes
Number
of Kernels
Expected
Number
Genotypes
of Kernels
Phenotypes will vary depending on the trait studied
Ears bearing F, kernels can be purchased from a number of biological supply companies. Without
removing the kernels from the ears, you can count the number of kernels in the different phenotypes,
record data in Table 1.3, and then formulate the hypotheses to explain the data collected. Two pos-
sible illustrations of endosperm traits follow
F2
I SUSHI
Parents
1 SUSH
X SUSU
starchy
sweet
(smooth) (wrinkled)
2. CC X сс
pigmented nonpigmented
(purple) (white)
F,
Susu
starchy
(smooth)
3 Su-
starchy
(smooth)
3 C-
pigmented
(purple)
Sweet
(wrinkled)
: lcc
nonpigmented
(white)
pigmented
(purple)
A host of other endosperm mutants have been researched. Many are able for classroom labo-
ratory investigation. Examples include Wx (starchy) versus wx (waxy), Y (yellow) versus y (white),
and Pr (purple) versus pr (red) endosperm. Franks (1980, 1981) and Neuffer et al. (1997) provide
further information and excellent color photographs of useful maize mutants.
VII. INDEPENDENT ASSORTMENT
A. Dihybrid Cross with Drosophila
Under the direction of the instructor, prepare a mating between flies carrying homozygous mutant
genes in the second and third chromosomes. Produce Fi and F2 generations. Several crosses are
appropriate for this experiment. Although one such cross will be suggested here, your instructor may
decide to use alternative crosses.
1. The gene for vestigial wings (vg) is located on the second chromosome, and the gene for ebony
body (@) color is located on the third chromosome. Matings may be made between flies from
these two stocks. Be certain to use virgin female flies for this cross. Reciprocal crosses, that is,
vestigial female (ugog; c'e') x ebony male cug'ug'; ee) and ebony female X vestigial male, may
be made if time and equipment permit. After about 8 days, remove and discard the parent flies.
Record all data relative to this experiment in Table 1.4.
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AG TT GGTC (GG 104T GG A7
а!
2 AGC 77 4G LTC64 7CaAcac AT
icar
7 (CCC (04ATTG7AATA
1
2 CCCCAGGAATTGTAATA
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