Download Example - Hivebench

Hivebench
Example
Created by noelsilvadom
BIO 502 Lab (Spring, 2014)
04/10/14
(Techniques in Genomics & Proteomics)
DESIGNING PRIMERS
EXPERIMENT OBJECTIVE: To design primers for cloning human Rheb1, Rheb2 and TCTP into
a mammalian expression vector (pCMV-BICEP4).
PRINCIPLE: A primer is a short synthetic oligonucleotide which is used in many molecular
techniques from PCR to DNA sequencing. These primers are designed to have a sequence
which is the reverse complement of a region of template or target DNA to which we wish the
primer to anneal.
When designing primers for PCR, sequencing or mutagenesis it is often necessary to make
predictions about these primers, for example melting temperature (Tm) and propensity to
form dimers with itself or other primers in the reaction.
Primer length and sequence are of critical importance in designing the parameters of a
successful amplification: the melting temperature of DNA duplex increases both with its
length, and with increasing (G+C) content: a simple formula for calculation of the Tm is
Tm = 4(G + C) + 2(A + T) oC.
Thus, the annealing temperature chosen for a PCR depends directly on length and
composition of the primer(s). One should aim at using an annealing temperature (Ta) about
5oC below the lowest Tm of the pair of primers to be used. One consequence of having too
low a Ta is that one or both primers will anneal to sequences other than the true target, as
internal single-base mismatches or partial annealing may be tolerated: this is fine if one
wishes to amplify similar or related targets; however, it can lead to "non-specific"
amplification and consequent reduction in yield of the desired product, if the 3'-most base
is paired with a target.
A consequence of too high a Ta is that too little product will be made, as the likelihood of
primer annealing is reduced; another and important consideration is that a pair of primers
with very different Tas may never give appreciable yields of a unique product, and may
also result in inadvertent "asymmetric" or single-strand amplification of the most efficiently
primed product strand.
Annealing does not take long: most primers will anneal efficiently in 30 sec or less, unless
the Ta is too close to the Tm, or unless they are unusually long.
The optimum length of a primer depends upon its (A+T) content, and the Tm of its partner if
one runs the risk of having problems such as described above. Apart from the Tm, a prime
consideration is that the primers should be complex enough so that the likelihood of
annealing to sequences other than the chosen target is very low.
For example, there is a ¼ chance (4-1) of finding an A, G, C or T in any given DNA sequence;
there is a 1/16 chance (4-2) of finding any dinucleotide sequence (e.g. AG); a 1/256 chance of
finding a given 4-base sequence. Thus, a sixteen base sequence will statistically be present
only once in every 416 bases (=4 294 967 296 or 4 billion): this is about the size of the
human or maize genome, and 1000x greater than the genome size of E. coli. Thus, the
association of a greater-than-17-base oligonucleotide with its target sequence is an extremely
sequence-specific process, far more so than the specificity of monoclonal antibodies in binding
to specific antigenic determinants. Consequently, 17-mer or longer primers are routinely
used for amplification from genomic DNA of animals and plants. Too long a primer length
may mean that even high annealing temperatures are not enough to prevent mismatch pairing
and non-specific priming.
1. Primer Length: It is generally accepted that the optimal length of PCR primers is 18-22 bp.
This length is long enough for adequate specificity, and short enough for primers to bind easily
to the template at the annealing temperature.
2. Melting Temperature: Melting Temperature (Tm) by definition is the temperature at which
one half of the DNA duplex will dissociate to become single stranded and indicates the duplex
stability. Primers with melting temperatures in the range of 52-58 oC generally produce the
best results. Primers with melting temperatures above 65oC have a tendency for secondary
annealing. The GC content of the sequence gives a fair indication of the Tm.
3. Primer annealing temperature: The primer melting temperature is the estimate of the
DNA-DNA hybrid stability and critical in determining the annealing temperature. Too high Ta
will produce insufficient primer-template hybridization resulting in low PCR product yield.
Too low Ta may possibly lead to non-specific products caused by a high number of base pair
mismatches. Mismatch tolerance is found to have the strongest influence on PCR specificity.
Ta = 0.3 x Tm (primer) + 0.7 Tm (product) – 14.9
where,
Tm (primer) = Melting Temperature of the primers
Tm (product) = Melting temperature of the product
4. GC Content: The GC content (the number of G's and C's in the primer as a percentage of the
total bases) of primer should be 40-60%.
5. GC Clamp : The presence of G or C bases within the lat five bases from the 3' end of primers
(GC clamp) helps promote specific binding at the 3' end due to the stronger bonding of G and C
bases. More than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the primer.
6. Secondary Structures: Presence of the secondary structures produced by intermolecular
or intramolecular interactions can lead to poor or no yield of the product. They adversely
affect primer template annealing and thus the amplification. They greatly reduce the
availability of primers to the reaction.
i) Hairpins: It is formed by intramolecular interaction within the primer and should be
avoided.
ii) Self Dimer: is formed by intermolecular interactions between the two (same sense)
primers, where the primer is homologous to itself. Generally a large amount of primers are
used in PCR compared to the amount of target gene. When primers form intermolecular
dimers much more readily than hybridizing to target DNA, they reduce the product yield.
iii) Cross Dimer: Cross dimers are formed by intermolecular interaction between sense and
antisense primers, where they are homologous.
7. Repeats: A repeat is a di-nucleotide occurring many times consecutively and should be
avoided because they can misprime. For example: ATATATAT. A maximum number of dinucleotide repeats acceptable are 4 di-nucleotides.
8. Runs: Primers with long runs of a single base should generally be avoided as they can
misprime. For example, AGCGGGGGATGGGG has runs of base 'G' of value 5 and 4. A maximum
number of runs accepted are 4bp.
Target genes: Human Rheb1, Rheb2 and TCTP
Cloning Vector: pCMV-BICEP-4
•
•
•
•
•
•
•
•
Obtain mRNA sequence of the gene listed above (explore NCBI)
Determine the coding sequence and highlight it
Look at the vector sequences and analyze the multiple cloning sites VERY CAREFULLY
Determine suitable sites into which the gene can be cloned (you will have to do a
restriction enzyme map of each of the gene using a program like NEB cutter)
Find RE sites that do NOT cut the gene- you will use these for cloning into the vector
Design the forward (5’) and reverse (3’) primers for the gene (keeping in mind the length
and GC content of each primer)- remember to incorporate the RE sites in the primers for
cloning; ENSURE THAT THE READING FRAME IS CORRECT
Submit primer sequences to me (Primers will be ordered from Invitrogen)
Once primers are received, proceed with PCR to amplify the gene