Polymerase Preference Index

• US 20120303283A1
• Filed: 01/19/2012
• Published: 11/29/2012
• Est. Priority Date: 01/19/2011
• Status: Active Grant

• Current Assignee: Irepertoirem Inc.
• Original Assignee: Irepertoirem Inc.
• Inventors: Han, Jian
• Granted Patent: US 10,860,684 B2
• Time in Patent Office: Days
• US Class Current: 702/19
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search
• CPC Class Codes: G16B 50/00 ICT programming tools or database systems specially adapted for bioinformatics

Abstract

Disclosed is a method for calculating a Polymerase Preference Index (PPI) for potential primers for DNA sequencing and/or amplification, and thereby increasing the efficiency of DNA sequencing and/or amplification performed using primers selected according to their PPI number.

Claims

1. A method comprising calculating a Polymerase Preference Index (PPI) using Equation I:

PPI=A/B*C/D*100 

 

(Equation I)

where A/B represents the probability of a 6-mer being used as a primer and C/D represents the probability of a 4-mer being used as a runway sequence.

2. A method comprising calculating a Polymerase Preference Index (PPI) using Equation II:

PPI=A/B*C/D* 

 

(Equation II)

where A/B represents the probability of a 6-mer being used as a primer and C/D represents the probability of a 4-mer being used as a runway sequence.

3. A computer-assisted method for identifying primers for polynucleotide amplification, the method comprising

(a) using a computer link to an internet site containing a program for performing the method, entering at least one target sequence for which one or more primer sequences are desired by a user;

(b) performing a search of one or more DNA sequence databases to identify one or more sequences having a desired sequence similarity to that of the target sequence;

(c) aligning the one or more sequences to identify one or more conserved regions within the target sequence;

(d) applying one or more analysis windows over the target sequence and applying Equation I

PPI=A/B*C/D*100 

 

(Equation I)

where A/B represents the probability of a 6-mer being used as a primer and C/D represents the probability of a 4-mer being used as a runway sequence, to one or more individual 10-nucleotide sequences within the target sequence; and

(e) identifying one or more potential amplicon regions, with a desirable potential amplicon being defined as a section of DNA of from about 100 to about 250 base pairs, where primer pairs, each primer being approximately 20 base pairs, are identifiable.

4. The method of claim 3, further comprising the step of (f) eliminating potential primer sequences likely to form secondary structures such as hairpin loops and primer dimmers.

5. The method of claim 4, further comprising the step of (g) balancing the results to obtain one or more sets with similar PPIs and to increase or decrease the length of selected primers to identify primers with similar melting temperatures.