GENETIC ENGINEERING I – SICKLE CELL DISEASE

Manipulating Recombinant DNA: Restriction Mapping,

Cloning, and PCR Primer Design

R estriction enzymes are sophis-

ticated “scissors” that molecu-
lar biologists use to cut DNA,
and they are routinely used in genetics
and molecular biology laboratories for
Copy the HBB coding sequence shown
below and paste it into ApE.

Human HBB CDS

ATGGTGCATCTGACTCCTGAGGA
1. On the second page is a plasmid DNA
sequence. Copy this sequence into a
new window in ApE, toggle the
linear/circular button to circular, and
identify cutting sites for the same
recombinant DNA experiments. Yet enzymes you used in Exercise I. Then
GAAGTCTGCCGTTACTGCCCTGT
another advantage of the genomics answer the following questions:
GGGGCAAGGTGAACGTGGATGA
revolution has been the development of a
AGTTGGTGGTGAGGCCCTGGGCA a. What is the total size of the plas-
wide variety of tools to assist scientists
GGCTGCTGGTGGTCTACCCTTGG mid DNA analyzed in ApE? Why did
working with restriction enzymes and
ACCCAGAGGTTCTTTGAGTCCTT you toggle to circular?
manipulating recombinant DNA for
TGGGGATCTGTCCACTCCTGATG
different applications, such as restriction b. Which enzyme(s) could be used in
CTGTTATGGGCAACCCTAAGGTG
mapping, designing primers for PCR a recombinant DNA experiment to
AAGGCTCATGGCAAGAAAGTGCT
experiments, and DNA cloning. Here we ligate the plasmid to the largest DNA
CGGTGCCTTTAGTGATGGCCTGG
explore ApE and Primer3, two tools that fragment from the human gene? Brief-
CTCACCTGGACAACCTCAAGGGC
make recombinant DNA experiments ly explain your answer.
ACCTTTGCCACACTGAGTGAGCT
much easier.
GCACTGTGACAAGCTGCACGTGG c. What size recombinant DNA mol-
Exercise I – Creating a Restriction ATCCTGAGAACTTCAGGCTCCTG ecule will be created by ligating these
Map in ApE GGCAACGTGCTGGTCTGTGTGCT fragments?
GGCCCATCACTTTGGCAAAGAAT
Suppose you had cloned and sequenced d. Draw a simple diagram showing the
TCACCCCACCAGTGCAGGCTGCC
the hemoglobin beta (HBB) gene and wanted cloned DNA inserted into the plasmid
TATCAGAAAGTGGTGGCTGGTGT
to design a roughly 300bp long probe that and indicate the restriction enzyme
GGCTAATGCCCTGGCCCACAAGT
could be used to analyze expression of cutting site(s) used to create this
ATCACTAA
this gene in different human tissues by recombinant plasmid. Try to clone in
Northern blot analysis. Not too long ago, 2. In the ApE toolbar, choose “Enzymes” ApE using “Tools” and Restriction-
you had primarily two ways to approach and “Enzyme selector”. A new window Ligation Assembler.
this task. You could digest the cloned will open, select the enzymes
DNA with whatever restriction enzymes mentioned before and click below on
were in your freezer, then run agarose “Digest”.
gels and develop restriction maps in the
hope of identifying cutting sites that 3. After examining the digestion results
would give you the size fragment you provided by ApE, describe what you
wanted. Or you could scan the sequence see.
with your eyes, looking for restriction
sites of interest— a very time-consuming Exercise II – Designing a
and eye-straining effort! Bioinformatic Recombinant DNA
tools such as ApE take the guesswork Experiment
out of developing restriction maps and Now that you have created a restriction
make it relatively easy to design map of your HBB CDS, you need to
experiments for manipulating ligate the DNA into a plasmid DNA
recombinant DNA. In this exercise, you vector that you can use to make your
will use ApE to create a restriction map probe (molecular biologists often refer to
with the enzymes BamHI and EcoRI. this as subcloning). To do this, you will
1. https://jorgensen.biology.utah.edu/way need to determine which restriction
ned/ape/ Go to the site, download and enzymes would best be suited for cutting
install ApE, and open it. both the plasmid and the human DNA.
 CGTTGGGAACCGGAGCTGAATGAAGCCAT annealing—or having primers bind to each
Plasmid DNA sequence
ACCAAACGACGAGCGTGACACCACGATGC other—among many other consider-
TATAAATATAGAATAATGAATCATATAA CTGTAGCAATGCCAACAACGTTGCGCAAA ations.
AACATATCATTATTCATTTATTTACATTT CTATTAACTGGCGAACTACTTACTCTAGCT
Fortunately, primer design is another task
AAAATTATTGTTTCAGTATCTTTAATTTA TCCCGGCAACAATTAATAGACTGGATGGA
made much easier by the Internet. In this
TTATGTATATATAAAAATAACTTACAATT GGCGGATAAAGTTGCAGGACCACTTCTGC
exercise, you will use Primer3, a PCR primer
TTATTAATAAACAATATATGTTTATTAAT GCTCGGCCCTTCCGGCTGGCTGGTTTATT
design site from the Whitehead Institute
TCATGTTTTGTAATTTATGGGATAGCGA GCTGATAAATCTGGAGCCGGTGAGCGTGG
for Biomedical Research.
TTTTTTTTACTGTCTGTATTTTTCTTTTTT GTCTCGCGGTATCATTGCAGCACTGGGGC
AATTATGTTTTAATTGTATTTTATTTTTA CAGATGGTAAGCCCTCCCGTATCGTAGTTA 1. Access Primer3 at http://bioinfo.ut.ee/
TTATTGTTCTTTTTATAGTATTATTTTAA TCTACACGACGGGGAGTCAGGCAACTATG primer3/. Copy the human DNA
AACAAAATGTATTTTCTAAGAACTTATA GATGAACGAAATAGACAGATCGCTGAGAT sequence from Exercise I into the text
ATAATAATAAATATAAATTTTAATAAAAA AGGTGCCTCACTGATTAAGCATTGGTAACT box, then click “Pick Primers.”
TTATATTTATCTTTTACAATATGAACATA GTCAGACCAAGTTTACTCATATATACTTTA
AAGTACAACATTAATATATAGCTTTTAA GATTGATTTAAAACTTCATTTTTAATTTAAA 2. On the next page, the sequences for
TATTTTTATTCCTAATCATGTAAATCTTA AGGATCTAGGTGAAGATCCTTTTTGATAAT the best recommended primers will
AATTTTTCTTTTTAAACATATGTTAAATA CTCATGACCAAAATCCCTTAACGTGAGTTT appear at the top of the screen. An-
TTTATTTCTCATTATATATAAGAACATAT TCGTTCCACTGAGCGTCAGACCCCGTAGA swer the following:
TTATTAAATCTAGAATTCTATAGTGAGT AAAGATCAAAGGATCTTCTTGAGATCCTTT a. What is the length, in base pairs, of
CGTATTACAATTCACTGGCCGTCGTTTT TTTTCTGCGCGTAATCTGCTGCTTGCAAAC the left (forward) primer and right (re-
ACAACGTCGTGACTGGGAAAACCCTGG AAAAAAACCACCGCTACCAGCGGTGGTTT verse) primer? Where does each of these
CGTTACCCAACTTAATCGCCTTGCAGC GTTTGCCGGATCAAGAGCTAC primers bind in the gene sequence?
ACATCCCCCTTTCGCCAGCTGGCGTAA
As you prepare to carry out this sub- b. The hybridization temperature for
TAGCGAAGAGGCCCGCACCGATCGCC
cloning experiment, you find that the a PCR reaction is often set around
CTTCCCAACAGTTGCGCAGCCTGAATG
expiration dates on most of your 5 degrees below the melting temper-
GCGAATGGCGCCTGATGCGGTATTTTC
restriction enzymes have long since ature, or Tm (refer to Chapter 10 for
TCCTTACGCATCTGTGCGGTATTTCACA
passed. Rather than run an experiment a discussion of melting temperature).
CCGCATATGGTGCACTCTCAGTACAAT
with old enzymes, you decide to purchase Based on the Tm for these primers,
CTGCTCTGATGCCGCATAGTTAAGCCA
new enzymes. Fortunately, a site called what might be the optimal hybridiza-
GCCCCGACACCCGCCAACACCCGCTG
REBASE®: The Restriction Enzyme tion temperature for this experiment?
ACGCGCCCTGACGGGCTTGTCTGCTCC
Database can help you. Over 300
CGGCATCCGCTTACAGACAAGCTGTGA c. What size PCR product would you
restriction enzymes are commercially
CCGTCTCCGGGAGCTGCATGTGTCAGA expect these primers to generate if you
available rather inexpensively, but
GGTTTTCACCGTCATCACCGAAACGCG ran the DNA amplified by this PCR re-
scientists are always looking for ways to
CGAGACGAAAGGGCCTCGTGATACGCC action on an agarose gel?
stretch their research budgets as far as
TATTTTTATAGGTTAATGTCATGATAATA
possible. REBASE is excellent for
ATGGTTTCTTAGACGTCAGGTGGCACTT
locating enzyme suppliers and enzyme
TTCGGGGAAATGTGCGCGGAACCCCTA
specifics, particularly if you need to
TTTGTTTATTTTTCTAAATACATTCAAAT
work with an enzyme that you are
ATGTATCCGCTCATGAGACAATAACCCT
unfamiliar with. Visit REBASE® at
GATAAATGCTTCAATAATATTGAAAAAG
http://rebase.neb.com/rebase/
GAAGAGTATGAGTATTCAACATTTCCGT
rebase.html to identify companies that
GTCGCCCTTATTCCCTTTTTTGCGGCAT
sell the restriction enzyme(s) you need
TTTGCCTTCCTGTTTTTGCTCACCCAGA
for this experiment. Click on one of the
AACGCTGGTGAAAGTAAAAGATGCTGA
companies and search for the enzyme
AGATCAGTTGGGTGCACGAGTGGGTTA
EcoRI. How expensive is it?
CATCGAACTGGATCTCAACAGCGGTAA
GATCCTTGAGAGTTTTCGCCCCGAAGA Exercise III – Designing PCR Primers
ACGTTTTCCAATGATGAGCACTTTTAAA
Giving this experiment more thought,
GTTCTGCTATGTGGCGCGGTATTATCC
you decide to try reverse transcriptase
CGTATTGACGCCGGGCAAGAGCAACTC
PCR (RT-PCR) first instead of
GGTCGCCGCATACACTATTCTCAGAAT
Northern blotting because RT-PCR is a
GACTTGGTTGAGTACTCACCAGTCACA
faster and more sensitive way to detect
GAAAAGCATCTTACGGATGGCATGACA
gene expression. Picking correct
GTAAGAGAATTATGCAGTGCTGCCATA
primers for a PCR experiment is not a
ACCATGAGTGATAACACTGCGGCCAAC
trivial process. You have to be sure the
TTACTTCTGACAACGATCGGAGGACCG
primers can amplify the gene of interest,
AAGGAGCTAACCGCTTTTTTGCACAAC
and you need to avoid primer self-
ATGGGGGATCATGTAACTCGCCTTGAT