John M. S. Bartlett.pdf - Bio-Nica.info

Cloning Gene Family Members 491
four nucleotides, creating a single bond in all cases (10). The disadvantage is that inosines
reduce the annealing temperature of the primer. I have not used inosine-containing primers
in my studies.
4. It is often convenient to incorporate restriction endonuclease sites at the 5′-ends of a primer
to facilitate cloning into plasmid vectors (4,8,9). Different restriction sites can be added
to the 5′-ends of different primers so the products can be cloned directionally. However,
not all restriction enzymes can recognize cognate sites at the ends of a double-stranded
DNA molecule equally well. This difficulty can often be reduced by adding a two to four
nucleotide 5′-overhang before the beginning of the restriction enzyme site (see Note 5).
Some of the best restriction enzymes sites to use are EcoRI, BamHI, and XbaI. Catalogs
from New England Biolabs have a list of the ability of different restriction enzymes to
recognize short base-pair sequences. A potential pitfall of this approach would be the
occurrence of the same restriction site within the amplified product as used on the end of
one of the primers. Therefore, only part of the amplified product would be cloned.
5. The final consideration you should make is the identity of the 3′ most nucleotide. The
nucleotide on the 3′-end of a primer should preferably be G or C and not N, I, or T.
The reason for this is that thymidine (and supposedly inosine) can nonspecifically prime
on any sequence. Guanosines and cytidine are preferred since they form three H-bonds at
the end of the primer, a degree stronger than an AT base pair.
3.2. PCR Amplification and DNA Purification
The template for these reactions can be the DNA in a phage library or the first-strand
cDNA from a reverse transcription reaction on RNA. A phage library with a titer of
5 × 10 9 pfu/mL would contain, in a 5-µL aliquot, 2.5 × 10 7 pfu (~1.5 ng of DNA).
Before PCR amplification, the DNA is heat denatured at 99°C for 10 min.
3.2.1. PCR (see Notes 1 and 6)
In all cases, the DNA template should also be PCR amplified with the individual
degenerate primers to determine whether any of the bands amplified are derived from
one of the degenerate primer pools. A DNA-free control is required to assess if there is
contaminating DNA in any of the other reagents.
1. Pipet into 0.5-mL microcentrifuge tubes in the following order: 58.5 µL of PCR-water that
has been autoclaved; 10 µL of 10× PCR buffer (see Note 2); 16 µL of 1.25 mM dNTP stock
solution; 5.0 µL of primer up-1; 5.0 µL of primer down-1; and 5.0 µL of heat-denatured
library or cDNA (1–100 ng). If several reactions are being set up concurrently, a master
reaction mix can be made up consisting of all the reagents used in all of the reactions, such
as the PCR water, reaction buffer, and dNTPs.
2. Briefly vortex each sample and spin for 10 s in a microfuge. Overlay each sample with
2 to 3 drops of mineral oil.
3. Amplify by hot-start PCR using the following cycle parameters. Pause the thermocycler
in step 4-cycle 1 and add 0.5 µL of Amplitaq DNA polymerase to each tube. Then, run for
95°C, 5 min (initial denaturation); 94°C, 60 s (denaturation); 50°C, 90 s (annealing; see
Note 7); 72°C, 60 s (extension); cycle 29 times to step 2; 72°C, 4 min; and 10°C hold.
3.2.2. DNA Isolation and Gel Electrophoresis Analysis
1. Remove the reaction tubes from the thermal cycler and add 200 µL of chloroform. Spin for
10 s in a microfuge to separate the oil-chloroform layer from the aqueous layer. Carefully
transfer the aqueous layer to a clean microfuge tube.