Computer Exercises
Weeks of September 18 and September 25, 2002
DATABASE SEARCH AND MULTIPLE SEQUENCE ALIGNMENT. These exercises will
be done the week of Wednesday, September 18. Remember, we are trying
to clone the gene encoding a protein called furin from cells (CHO-K1
cells) derived from hamsters. Where do we begin in this project?
Well, PCR technology will allow us to "amplify" at least a piece of
this gene if we could identify at least two DNA sequences likely to be
present in the CHO-K1 gene encoding furin. How can we identify such
DNA sequences? Quite easily, as it turns out. First, we will access
a DNA and protein sequence database called GenBank and obtain the
deduced amino acid sequences of furin proteins from several species.
The genes encoding furin proteins are called fur genes, and this
nomenclature will need to be used to access relevant sequences in the
database. Once we have obtained these sequences, we will download
them into a software package that will generate a multiple sequence
alignment, which essentially lines up all the protein sequences from
the amino to carboxy termini. The software is called Lasergene; it is
made by a company called DNASTAR (Madison, WI). DNASTAR has kindly
given us an Educational Use License, allowing us to utilize the
software in this class. The software package will not only align the
sequences for us, but will also generate what is called a consensus
sequence to show how conserved the amino acids are at each position in
the alignment; in other words, the consensus sequence will indicate
areas of amino acid identity--that is, areas where the amino acids in
all of the sequences are identical. This is the information we are
looking for. Why is this important? Well, furin amino acid sequences
that are evolutionarily conserved in the proteins whose sequences have
been deposited in the database are likely to be conserved in other
species as well--including, hopefully, hamsters. Considering what you
know about the relationship between the structure and function of
macromolecules, what might you conclude about the amino acids that are
conserved? If we are reasonably certain about the amino acid sequence
of a piece of hamster furin, we can also be reasonably sure about the
nucleotide sequence of the gene as well, and this will allow us to
design oligonucleotide primers to amplify a piece of the hamster fur
gene.
Instructions for accessing the database and generating the multiple
sequence alignment will be handed out prior to the laboratory session.
We will attempt to have a color copy of the alignment and consensus
sequence printed out for each individual, but this may not be possible
to do.
HOMEWORK FOR NEXT WEEK. Prior to next week's lab session, each of you
should take the alignment and 1) identify regions that you could use
to make primers to, and 2) determine the nucleotide sequence of your
primers. Keep in mind that you are looking for areas of amino acid
identity among all the furin proteins in the database, since these
conserved sequences are likely to be found in hamster furin as well.
There are several other things to keep in mind when selecting
sequences to design primers to. First, the smaller the region to be
amplified the better, to a point. We would ideally like to try to
amplify a piece of DNA ranging in size from 250 bp to 750 bp; this
would correspond to amino acid distances of about 80 to 250, so keep
this in mind when examining the alignment for conserved sequences.
Another thing to keep in mind when looking for sequences is that codon
redundancy exists for most amino acids. Look at the attached codon
preference chart for E. coli. Notice that serine, for example, has 6
different codons, and, at least in E. coli, there is no obvious
"consensus codon". Thus, a highly conserved amino acid sequence that
includes serine may not be a good choice to design primers to, and
this is especially true if the serine or serines are located such that
they would correspond to the 3' end of a primer; we will talk a little
more about this in class. However, to simplify your primer design,
your instructor has provided a list of "preferred hamster codons".
Once you have identified amino acid sequences you think would work for
primers, derive the nucleotide sequences for your primers (using the
preferred hamster codons), and keep in mind that each pair of primers
should have one primer annealing to each DNA strand. This is
something else we will talk about more in class.
SOFTWARE ANALYSIS OF THE PRIMERS. The week of Wednesday, September
25, you will hopefully come to lab with your primer sequences, and we
will use another aspect of the Lasergene software package to analyze
the primers for several things, as described below. Instructions for
doing these things will be given in the laboratory session.
1. The software will analyze your primers for the potential to form
stable secondary structures. The diagram below illustrates how an
oligonucleotide primer can fold into a stable "hairpin".
Primer Sequence: 5'-TTCATGCCGTATCCAATACGGC-3'
Secondary Structure: 5'-TTCATGCCGTAT
C
3'-CGGCATA
If a primer can form this type of secondary structure, it can result
in a competition between whether this structure forms or whether the
primer actually anneals to the target sequence, and, obviously, if you
are trying to do PCR this is not good!! When examining secondary
structures your primers may form, be sure to consider the free energy
of formation (∆G) values shown by the software; secondary structures
that are not energetically favorable will not likely hinder PCR
reactions. Another thing to consider is the melting temperature of
the structures (also called TM, which we will talk about). Even if a
structure can readily form, if it has a melting temperature below the
temperatures used in the PCR reactions, the primer can likely still be
used as the structure will not be stable (if it forms).
2. The software will also analyze your primers for the ability to
form what are called primer dimers; primer dimers form when the
primers or a primer has complimentary sequences at the termini, as
shown below:
Primer Sequence: 5'-CCTTTGCCCAGTCGTACGAC-3'
Primer Dimer: 5'-CCTTTGCCCAGTCGTACGAC-3'
3'-CAGCATGCTGACCCGTTTCC-5'
This diagram shows a primer dimer forming from two primers of the same
sequence; primer dimers can also form with two different primers, if
they have complimentary termini. Free energy of formation values and
melting temperatures should also be considered when deciding if primer
dimers are potentially detrimental for use.
3. The software will take your primers and, with any set of conditions
(i.e., primer concentration, salt concentration) will tell you where
on a specific template (in this case our furin consensus sequence,
derived from our amino acid sequence) your primers will bind. This
information will be helpful in deciding whether or not to use your
primers as designed; for example, if one of your primers is able to
bind to several different sites within the template DNA, you will not
want to use this primer as you will not likely be able to generate a
specific reaction product through PCR. However, doing this type of
analysis requires the sequence of the template. Also, when examining
these primer binding site, the ∆G numbers and melting temperatures
(between primer and template) should again be considered; if the
binding of the primer to a "non-specific" template sequence--that is,
a sequence other than where you want it to bind--is not energetically
favorable and/or has a low melting temperature, that primer may still
be able to be utilized for PCR.
4. You should also calculate the melting temperatures of the primers
at their target sequences. This will help us set up the parameters for
the subsequent PCR. Also, if the TM values are radically different
for these two primer-template annealing events (remember for PCR you
need two primers), it is likely to cause problems when trying to run
PCR reactions.
Based on all of the above criteria, can you theoretically use any of
the primers you have designed?
SUPPLEMENTAL INFORMATION:CODON PREFERENCES IN E.coli AND HAMSTERS*
The percentage of each codon for the indicated amino acids is shown
for E. coli (in black) and for hamsters (i.e. CHO-K1 cells) in green.
Leucine (L)
Serine (S) Arginine (R) Valine (V)
CTG 83% (8%) TCC 37% (10%) CGT 74% (15%) GTT 51% (5%)
CTC 7% (57%) TCT 34% (9%) CGC 25% (3%) GTA 26% (14%)
CTT 4% (6%) AGC 20% (18%) CGA 1% (1%) GTG 16% (59%)
TTG 3% (16%) TCG 4% (5%) AGG 0% (5%) GTC 7% (22%)
TTA 2% (3%) AGT 3% (48%) AGA 0% (4%)
CTA
0% (9%) TCA 2% (11%) CGG 0% (71%)
Glycine (G)
Threonine (T) Alanine (A) Isoleucine (I)
GGT 59% (69%) ACC 55% (60%) GCT 35% (7%) ATC 83% (68%)
GGC 38% (21%) ACT 35% (31%) GCA 28% (4%) ATT 17% (32%)
GGG 2% (6%) ACG 7% (6%) GCG 26% (51%) ATA 0% (0%)
GGA
0% (3%) ACA 4% (4%) GCC 10% (38%)
Aspartate (D)
Phenylalanine (F) Lysine (K) Tyrosine (Y)
GAC 67% (24%) TTC 76% (50%) AAA 74% (28%) TAC 75% (87%)
GAT 33% (76%) TTT 24% (50%) AAG 26% (72%)
TAT 25% (13%)
Proline (P)
Glutamate (E) Asparagine (N) Glutamine (Q)
CCG 77% (2%) GAA 78% (24%) AAC 94% (83%) CAG 86% (85%)
CCA 15% (18%) GAG 22% (76%) AAT 6% (17%) CAA 14% (15%)
CCT 8% (4%)
CCC 0% (76%)
Histidine (H)
Cysteine (C) Tryptophan (W) Methionine (M)
CAC 83% (59%) TGT 86% (92%) TGG 100% (100%) ATG 100% (100%)
CAT 17% (41%) TGC 14% (8%)
*The E.coli codon preferences are for "highly expressed genes"; this
information was compiled by Dr. Brian Foley of the University of
Vermont (Burlington, VT) and the Los Alamos National Laboratory (Los
Alamos, NM). The hamster codon preferences were compiled by Dr. Joe
Sucic and Holly Sucic, with assistance from the Incredible Talking
Hamsters: Emerson, Winslow, Drexel, and Avery.
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