MCB 150 Chapter 21-26: mcb150 - answer-key-chptr-21-26

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Published on 28 Jun 2016
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University of Illinois
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Molecular and Cell Biology
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MCB 150
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Notes to Instructors
Chapter 21 Genomes and Their Evolution
What is the focus of this activity?
While the Sanger method for sequencing DNA and the modifications that follow are
conceptually fairly simple, most students don’t understand them. As noted previously, in
order for most students to understand unfamiliar processes, they need to build models for
themselves to discover what it is they understand and, more important, what they don’t.
What is this particular activity designed to do?
Activity 21.1 How can we discover the sequence of an organism’s DNA?
This activity allows students to interpret the results of a classic Sanger method for
sequencing a DNA molecule only 20 bp long. It then asks them to translate these results
into the results they would expect using more modern fluorescently tagged ddNTP
methods.
What misconceptions or difficulties can this activity reveal?
Because much of the methodology and information in this area is relatively new, students
tend to lack conceptions in this area rather than have misconceptions. Walking them
through how sequencing is done and how it is interpreted should overcome this.
Answers
Activity 21.1 How can we discover the sequence of an
organism’s DNA?
Bacterial genomes have between 1 million and 6 million base pairs (Mb). Most plants and
animals have about 100 Mb; humans have approximately 2,900 Mb. Individual
chromosomes may therefore contain millions of base pairs. It is difficult to work with
DNA sequences this large, so for study purposes the DNA is broken into smaller pieces
(approximately 500 to 1,000 bp each). These pieces are sequenced and then the
sequenced pieces are examined and aligned based on overlapping sequence homology at
their ends.
144 Notes to Instructors
Copyright © 2011 Pearson Education, Inc.
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Activity 21.1 145
Copyright © 2011 Pearson Education, Inc.
By comparing the DNA sequences among organisms, scientists can determine
what parts of the genomes are most similar among organisms and are therefore
likely to have evolved earliest,
what key differences exist in the genomes that may account for variations among
related species, and
what differences within species exist that may account for development of
specific types of disease.
The following activity has been designed to help you understand how genomes are
sequenced and how the sequence information may be used.
1. In 1980, Frederick Sanger was awarded the Nobel Prize for inventing the dideoxy
method (or Sanger method) of DNA sequencing. A double-stranded DNA segment
approximately 700 bp in length is heated (or treated chemically) to separate the
two strands. The single-stranded DNA that results is placed into a test tube that
contains a 9-to-1 ratio of normal deoxynucleotides to dideoxynucleotides. A
dideoxynucleotide has no OH group at either 2or 3carbon. As a result,
whenever any dideoxynucleotide (abbreviated ddNTP) is added to the growing DNA
strand, synthesis stops at that point. If the ratio of normal to dideoxynucleotides is
high enough, where the dideoxynucleotide (rather than the normal deoxynucleotide)
will be included in the sequence is random.
You set up each of four test tubes as noted below:
All tubes contain the same single-stranded DNA molecules and the same primers. All
other components required for DNA replication, such as enzymes, are present in each
tube. You allow the replication to continue for the same length of time in each tube. At the
end of the time period, you extract the DNA from each tube and run it on an agarose gel.
You dye the gel with ethidium bromide and observe the following banding patterns on the
gel. (Note: For this demonstration, we are using a DNA strand that is only 20 bases in
length.)
Tube number Deoxynucleotides Dideoxynucleotide
1dATP, dTTP, dGTP, dCTP ddATP
2dATP, dTTP, dGTP, dCTP ddTTP
3dATP, dTTP, dGTP, dCTP ddGTP
4dATP, dTTP, dGTP, dCTP ddCTP
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146 Activity 21.1
Copyright © 2011 Pearson Education, Inc.
a. Which band in the gel contains the shortest DNA strand? What is the identity of
its terminal ddNTP?
The last band in the ddCTP column contains the shortest DNA strand. (Remember,
the shorter the DNA the faster it moves in the gel.) The terminal ddNTP is C.
b. Which band contains the next shortest DNA strand? What is the identity of its
terminal ddNTP?
The last band in the ddTTP column is the next shortest. The terminal ddNTP
here is T.
c. Continue reading the terminal ddNTP of each band from shortest to longest to
determine the linear sequence of nucleotides in the DNA strand complement.
What is the sequence?
The sequence (reading from shortest to longest) would be:
CTGCTTAATCGTATGCGATT.
2. The Sanger method has been modified so that each ddNTP used is now flagged with
an identifying fluorescent tag.
Assume that you run the same experiment that you did earlier. However, this time
you combine all of the different nucleotides (both dNTPs and ddNTPs) in the same
test tube. You run the products of the reaction on an agarose gel. Indicate the bands
you would see on the gel below using the appropriate colors: ddTTP fluoresces red;
ddGTP, yellow; ddCTP, blue and ddATP, green.
ddATP tube ddTTP tube ddGTP tube ddCTP tube
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Document Summary

While the sanger method for sequencing dna and the modifications that follow are conceptually fairly simple, most students don"t understand them. As noted previously, in order for most students to understand unfamiliar processes, they need to build models for themselves to discover what it is they understand and, more important, what they don"t. This activity allows students to interpret the results of a classic sanger method for sequencing a dna molecule only 20 bp long. It then asks them to translate these results into the results they would expect using more modern fluorescently tagged ddntp methods. Because much of the methodology and information in this area is relatively new, students tend to lack conceptions in this area rather than have misconceptions. Walking them through how sequencing is done and how it is interpreted should overcome this. Bacterial genomes have between 1 million and 6 million base pairs (mb).