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MOLECULAR BIOLOGY: WORKING WITH DNA
POLYMERASE CHAIN REACTION
GENERATION OF A PROBE FOR cDNA CLONING BY PCR USING PEPTIDE SEQUENCE INFORMATION
Generation of a Probe for cDNA Cloning by PCR Using Peptide Sequence Information
This protocol describes the design of DNA oligonucleotides from peptide sequence information and a "touchdown" PCR protocol for subsequent PCR amplification of the target cDNA. Procedure
1. After obtaining the amino acid sequence from two poly-peptides of interest, design two DNA oligonucleotide primers for each peptide (this is a total of four primers for two top choice peptides). The two primers should be 5' to 3' in the coding strand and 5' to 3' in the non-coding strand of the DNA corresponding to the peptide sequence since nucleotides are only added to the 3' end by Taq DNA polymerase (see Hint #1). Use the following general considerations in the oligonucleotide design:
a. Use the codon bias of the organism to select only those nucleotides that will be present in a multiple codon three nucleotide sequence.
b. Use the Inosine nucleotide in cases in which the degeneracy is high. Inosine will basepair with all four nucleotides and does not add or subtract from the overall calculation of melting temperature.
c. Try to minimize the degeneracy of the primers while maintaining a melting temperature greater than 35°C and with enough G and C content.
d. If convenient for future cloning steps, add a sequence to the 5' end of each oligonucleotide corresponding to the recognition site for a restriction enzyme of your choice. Consult Citation #1 or a manufacturer's catalogue for information on how many nucleotides are required for recognition of a restriction site by a particular enzyme.
2. Prepare four PCR reactions for the combinations of pairs of each of the primers by using a cDNA library or equivalent source of DNA (i.e., directly reverse transcribed cDNA from mRNA) for the template in the reaction:
Add the following to each reaction mixture:
50 to 100 ng of cDNA library DNA or equivalent
10 μl of 10X Taq DNA Polymerase Buffer
Add ddH2O to 100 μl
200 μM dNTPs (final concentration)
1 μM of each Primer in a pair
2.5 Units of Taq DNA Polymerase
3. Calculate the initial Melting temperature (Tm) of each primer according to the following rules:
Each T or A is equivalent to 2°C
Each G or C is equivalent to 4°C
Each I is equivalent to 0°C
4. Based on the calculated Tm (see Hint #2), program the PCR machine with the following profile:
Begin at 94°C for 2 min
Two cycles at:
94°C for 1 min
(x)°C for 1 min, where (x) is 1°C above calculated Tm
72°C for 1 min
Repeat the above cycling profile with the following changes: Lower the annealing temperature 1°C every 2 cycles until the annealing temp has reached 6°C below the Tm.
Then run 25 cycles of the following profile:
94°C for 1 min
(x)°C for 1 min, where (x) is the Tm
72°C for 1 min
Finish the PCR at 72°C for 5 min
5. Analyze one-tenth of the reaction on an Agarose or Acrylamide gel (see Protocol ID#1265).
6. Isolate the PCR products from the gel (see Protocol ID#122).
7. Digest the PCR products with the appropriate restriction enzymes and clone the products in the appropriate vector (see Hint #3).
8. Sequence the clones to identify the correct PCR product.
9. The correct clone can now be propagated and used as a probe (see Protocol ID#9024).
Taq DNA Polymerase See Step #2 of the protocol
2.5 Units/reaction of Taq DNA Polymerase
1 M dNTP See Step #2 of the protocol
1 M dATP
The final concentration of dNTPs in the PCR mix is 200 μM
1 M dGTP
1 M dTTP
1 M dCTP
Taq DNA Polymerase Buffer (10X) Supplied by the manufacturer of Taq DNA Polymerase cDNA Library Or an equivalent source of DNA Peptide Primer #2 For the peptide sequence #2
Primer 4: 5' to 3' non-coding strand
Primer 3: 5' to 3' coding strand
Peptide Primer #1 Primer 2: 5' to 3' non-coding strand
Primer 1: 5' to 3' coding strand
For the peptide sequence #1
BioReagents and Chemicals
DNA Polymerase, Taq
1. Consult with standard references that describe degenerate oligonucleotide design (Molecular Cloning: A laboratory manual, p. 11.17-11.18, Current Protocols, etc.).
2. If the oligonucleotides differ in Tm, choose the Tm of the lower temperature oligonucleotide for the PCR conditions.
3. Choose a vector that contains restriction enzyme recognition sites corresponding to the sites present in the designed primers.
Citation and/or Web Resources
1. Molecular Cloning: A laboratory manual, Maniatis, Fritsch, and Sambrook (1982): p. 11.17-11.18. NAR (1991), 19: 4008.