ARRANGEMENT AND METHOD FOR THE ANALYSIS OF NUCLEOTIDE SEQUENCES

Abstract: The invention comprises the following functions which are incorporated in a handheld arrangement: means specifically adapted for manual input of nucleotide sequences, means for analyzing said sequences, means for displaying the secondary structure of said sequences and means for storing information. Optionally the arrangement comprises means for communication with other devices. Also, the invention comprises a method that makes use of the arrangement.

Description: Title Arrangement and method for the analysis of nucleotide sequences The present invention pertains to a handheld arrangement specially adapted for the analysis of nucleotide sequences, such as DNA, cDNA, RNA, PNA and oligonucleotide sequences and in particular a handheld arrangement, suitable for use in a laboratory environment, such as in a biotech laboratory. Also, it comprises a method making use of said arrangement. Background of the invention The handling of genetic information in the shape of nucleotide sequences has become routine work. Daily laboratory practice includes the analyzing of DNA, cDNA, RNA, PNA and oligonucleotide sequences to determine their annealing and melting temperatures, alignment with different sequences and secondary structure including possible overlapping. Presently this is done using cumbersome reference guides, available either in printed form of digitally, e.g. on CD- ROM or on databases. Certain databases are available on the Internet. The constant use of a PC or Macintosh-type computer in the laboratory is however not without its drawbacks. Presently, a person active in the field of biotechnology and/or genetic engineering works according to the following routines. Firstly, the skilled person may be studying a known DNA sequence and may want to extend said sequence. In order to retrieve further information, the practitioner has to design and manufacture one or several DNA primers. These are required to have certain properties in order to perform well in the PCR step (PCR is an acronym for Polymerase Chain Reaction). Examples of requirements, frequently encountered in these reactions are the following: the primer should have a CG-content of about 50 %, the bases should not be complimentary to each other in order to avoid the forming of so called hairpin loops or primer dimers. Further, the annealing temperature should be in the interval of between 45 and 60 °C. Usually, these questions are handled by using a stationary, normal computer with suitable software. Alternatively, this questions are clarified using pen and paper and consulting reference literature, available both in printed form and digitally, on CD-ROM and on the Internet. In doing these evaluations manually, there is always a considerable risk for errors. When the primer design is completed, the primer is manufactured or ordered from a suitable company or laboratory specializing in this field. After receiving the primer, the PCR- CONFIRMATION reaction is prepared, possibly again using some of the above steps to determine annealing temperature. There are, however, a number of algorithms available and the results may vary depending on which algorithm is used. The repeated and frequent use of a conventional computer or reference literature in the laboratory environment is cumbersome and awkward. There consequently remains a need for an user friendly, handheld device capable of the previously described functions. Summary of the invention The present invention makes available a handheld arrangement or device in accordance with the characterizing part of the independent claims 1 and 4. Other embodiments of the invention are described through the dependent claims. Such a device is well suited for use in a laboratory environment, specifically adapted for the analysis of nucleotide sequences and lacks the disadvantages associated with prior art devices, mainly personal computers, and prior art methods, mainly manual calculations and the consultation of reference manuals. As it is not possible to have a sufficient number of stationary computers or laptop computers in a laboratory due to test tubes and other fragile instruments and devices, the present invention solves related problems, in that it provides a small dedicated device for use in a "messy" laboratory environment. Also, when the PCR reaction is run on a PCR instrument, the present invention is easily used to verify a PCR parameter setting, which else would be accomplished with paper and pencil, thus introducing a possible source of error. Hence, the present invention allows a laboratory technician always to carry the device of the present invention, e.g. in a pocket in a hard environment where it is very easy to unintentionally destroy important material, such as test tubes and instruments. It is impossible to count on having a PC or even a laptop computer at one's disposal whenever needed in a laboratory. The fact that the device according to the present invention is dedicated to one task, namely to compute a primers PCR parameters, this with an adapted key set, thereby minimizing the sources of error, even excludes the use of smaller handheld computers such as palmtop computers. For the purpose of solving problems related to analysis of nucleotide sequences, the present invention introduces a handheld arrangement for the analysis of said nucleotide sequences, said arrangement comprising: input means specifically adapted for entering a nucleotide sequence corresponding to the four nucleotides A, C, G and T/U, thus minimizing possible sources of error by effecting a wrong input means; memory means for storing the entered information; means for analyzing the entered information; means for graphically displaying the secondary structure of an input sequence; and whereby said arrangement is available at every work space in a laboratory environment by carrying it, thus further further minimizing the risks of introducing sources of error. In one embodiment of the invention comprises means for transferring information wireless between said arrangement and other devices. Another embodiment of the arrangement comprises a timer. Further, the present invention comprises a method for the analysis of nucleotide sequences comprising the use of a handheld arrangement including. inputing a nucleotide sequence in said arrangement through input means corresponding to the four nucleotides A, C, G and T/U, thus minimizing possible sources of error by effecting a wrong input means; storing sequence in said arrangement; analyzing said sequence through said arrangement; graphically displaying a secondary structure of an input sequence through said arrangement; and whereby said arrangement is available at every work space in a laboratory environment by carrying it, thus further minimizing the risks of introducing sources of error. One embodiment of the method includes transferring information wireless between said arrangement and other devices. In another embodiment, the arrangement uses a timer. Brief description of the drawings The present invention will be described in closer detail below, with reference to the attached drawings, in which: Fig. 1 shows a schematic block diagram illustrating the device and means comprised by the same; Fig. 2 shows a frontal view of a device according to the invention; Fig. 3 shows a perspective view of the device in fig. 2; and Fig. 4 shows a frontal view of a device according to one embodiment of the invention. Detailed description of preferred embodiments It is appreciated that an arrangement and a method according to the present invention introduces safety measures minimizing sources of error by pressing the wrong keys on conventional computers. The arrangement also minimizes sources of error by being available in every spot of a laboratory, thereby being able of abandoning the use of paper and pencil when checking analysis made or to be made. As well known, the variable part of DNA is its sequence of bases. DNA contains four kinds of bases. The two purines are adenine (A) and guanine (G). The two pyrimidines are thymine (T) and cytosine (C). In RNA, thymine is substituted by uracil (U). A device, specifically adapted for the analysis consequently only needs to have means for entering this information and basic commands for performing the steps of analyzing the entered information. Regardless of this, presently available means employ a complete key board, typical for a conventional computer. This makes it practically impossible to down scale the device. A technician working in a laboratory preparing a PCR amplification of a DNA template is bound to design specific types of primers comprising short synthetic DNA fragments, so called oligonucleotide probes. Thereafter, such primers are manufactured by a molecular biologic services laboratory and delivered to the technician. When they are designed, the DNA string, the so called template, is to be amplified. At the distal ends of the template suitable areas are identified, where the primer is able to hybridize, i.e. adhere. Principals for hybridizing of oligonucleotide primers are, for example, described in J. Sambrook et al, "Molecular cloning, A Laboratory manual", second edition, pp 11.45-11.57. An analysis of a primer's properties is provided, by entering a primer sequence in a computer through its keyboard or by a handheld device or arrangement according to the present invention, wherewith parameters are computed. Examples of such parameters are annealing and/or melting temperatures and structure data for a primer. Supported by these parameters it is possible to select a suitable primer pair. In order to perform the PCR reaction, a dedicated program is assigned to the PCR instrument which provides temperatures cycles to be run by the instrument. To compute temperatures according to above, a DNA calculator is used presently a stationary computer. Character combinations of A, C, G, T, the DNA sequence, are representing the basic nucleotides comprised in the primer. The defined sequence is input to the calculator with the keys of the stationary computer keyboard with a great number of superfluous keys for the specific task. The computer then calculates and outputs the PCR reaction parameters. When the PCR parameter computing is finished, the PCR instrument is used whit the computed parameter setting. The PCR instrument is a well used resource in a laboratory, and therefore probably time scheduled for its use. Hence, it is useful to have access to a handheld device according to the present invention, described more in detail below. The present invention for the first time makes available a hand-held device which minimizes sources of error when computing PCR parameters, comprising the following means: a) input means 1 specially adapted for entering a nucleotide sequence, that is the four characters A, C, G and T or for RNA; A, C, G and U representing the four bases constituting DNA and RNA, respectively. Said input means can be implemented as depressable keys or pads, piezoelectric surfaces or touch sensitive areas on a display. Importantly, the number of input means is limited to four, corresponding to the four bases A, C, G and T U, supplemented by means for activating the device, such as an on-off switch and possible keys for selecting mode of operation. b) memory means 4] jfbr storing the entered information, such as conventional memory modules, for example a commercially available RAM-memory, c) means 2 for analyzing the entered information, such as conventional microprocessors, d) means 3 for graphically displaying the secondary structure of the input sequence, such as a conventional liquid crystal display (LCD) or similar display device, capable of a satisfactory resolution. Preferably the device according to the present invention further comprises e) means 5 for transferring information between said device and a stationary computer, e.g. IR-emitting and -registering means. The stationary computer in its turn can be part of a network, such as a local area network (LAN) which further can comprise connections to CD- ROM handling equipment, modems for access to the Internet, to mention only a few examples. Said means for transferring information between said device and a stationary computer can also be implemented as a docking station, enabling fast data transfer and simultaneous charging of the batteries in said handheld device according to the invention. Most preferably, the device according to the invention is implemented as a handheld network computer (NC), specifically adapted for nucleotide analysis. Further, the device according to the invention preferably comprises f) a timer for timing functions well known to a person skilled in the art of programming. By constructing a highly specialized device, eliminating an array of functions unnecessary for the analysis of nucleotide sequences, it is possible to make available a device with many advantages over prior art devices. The inventive device can be produced to a cost significantly lower than presently available computers, also compared to presently available handheld multipurpose computers, so called palm top computers. The inventive device is preferably made shock resistant and water resistant, thus better suited for use in a laboratory environment. Further, the greatly simplified keyboard makes the inventive device more user friendly and makes possible a speed of operation, for example entering of information, not possible using a conventional keyboard. A device according to the invention is put together using a conventional microprocessor, such as a commercially available type 386 processor or other comparable processor. Naturally, a more powerful processor can be used, such as a type 486 or 586, a so called Pentium ® -processor (Intel Inc.). The choice of hardware is optional within the scope of the invention and the most suitable hardware can be chosen by a skilled person, without departing from the scope or the invention. Suitable keys 9 are incorporated in the device, such as depressable keys or touch sensitive keys. Additional keys can be provided, such as an on-off switch and one or several keys for selecting the mode of operation ^ Further, a suitable display 7 is chosen, such as a LCD of suitable size and dissolution. Conventional electronic components are chosen by the skilled person, according to the requirements previously presented in order to assemble a shock resistant and water resistant device to a low cost of production. The device is finally given a casing or housing 6 suitable for the intended use, preferably an ergonomically adapted shape. The inventive device finally is given a casing with no dimension exceeding about 15 cm and preferably less than 10 x 10 cm, making it suitable to carry in a pocket, such as a breast pocket of a shirt or protective clothing normally used in a laboratory. Also the present invention introduces a method for the analysis of nucleotide sequences comprising the use of a handheld arrangement including: inputing a nucleotide sequence in said arrangement through input means corresponding to the four nucleotides A, C, G and T/U, thus minimizing possible sources of error by effecting a wrong input means; storing sequence in said arrangement; analyzing said sequence through said arrangement; graphically displaying a secondary structure of an input sequence through said arrangement; and whereby said arrangement is available at every work space in a laboratory environment by carrying it, thus further minimizing the risks of introducing sources of error. One embodiment of the method includes transferring information wireless between said arrangement and other devices. In another embodiment, the arrangement uses a timer for timing functions well known to a person skilled in the art of programming. The following pseudo code program listing provides one embodiment of a computing means for computation of PCR instrument input parameters, said parameters being computed by the arrangement, comprising said computing means, according to the present invention. States/modes defining the handheld arrangement So off state, implemented in hardware Si input state s 2 displaying state for computed parameters s, timer state s, scroll mode Events Ao Off key to off A, Off key to on A 2 Pressed key A A, Pressed key C A 4 Pressed key G A. Pressed key T Aβ Pressed key F, A 7 Pressed key F 2 A 8 Pressed key F 3 A, Pressed key F 4 State transitions and Function specifications S j : A, → S 0 turning off S 2 : Ao → S 0 turning off S 3 : Ao -» S 0 turning off S 4 : Ao → S 0 turning off S 0 : A, → ≤ ! power on Entering of data and parameter computations S,: A 2 → S, if strlen < 70 { append A to input string and display input string } else { display input string beep } Si: A 3 → S, if strlen < 70 { append C to input string and display input string } else { display input string beep } S t : A 4 → S, if strlen < 70 { append G to input string and display input string } else { display input string beep } S,: Aj → S, if strlen < 70 { append T to input string and display input string } else { display input string beep } S,: A 6 → S, if strlen > 0 { truncate last position in input string and display input string } else { display input string beep } S λ : A 7 -> S 2 compute parameters: L = strlen //Integer G/C% = 100*numberGC/L //One decimal if strlen < = 70 && strlen > 13 { compute parameters: T m = (81.5 + 0.41*numberGC) - (600/L) //One decimal T. = T m - 5 //One decimal } else { T m = T. = 0 } display parameter values Sj: A, - S 3 Timer is activated S^ A, -» S 4 Activate scroll mode Display computed parameters S 2 : A 2 -> S, Clear display and internal variables S 2 : A 3 - S, Clear display and internal variables S 2 : A 4 -> S, Clear display and internal variables S 2 : A 5 -» S, Clear display and internal variables S 2 : Aβ - S, Clear display and internal variables S 2 : A 7 - S, Clear display and internal variables S 2 : A, - S 3 Timer activated S 2 : A, → S, nop Timer: S 3 : A 2 → S 3 HH+ + , display HH:MM:SS S 3 : A 3 → S 3 MM+ + , display HH:MM:SS S 3 : A → S 3 SS+ + , display HH:MM:SS S 3 : A 5 → S 3 nop S 3 : A « → S 3 while HH:MM:SS > 0 { HH:MM:SS- - display HH:MM:SS } if HH:MM:SS = = 0 alarm else sleep 1 sec S 3 : A 7 → S, Return to initiahzed input mode S 3 : A 8 - S 3 nop S 3 : A, -> S 3 nop Scroll mode: S 4 : A 2 → S 4 if strlen < 70 && cunentPos < 70 { insert A in input string at cunentPos and display input string } else { display input string beep } S 4 : A 3 → S 4 if strlen < 70 && cunentPos < 70 { insert C in input string at cunentPos and display input string } else{ display input string beep } S 4 : A 4 → S 4 if strlen < 70 && cunentPos < 70 { insert G in input string at cunentPos and display input string } else { display input string beep } S 4 : A 3 - S 4 if strlen < 70 && cunentPos < 70 { insert T in input string vid cunentPos and display input string } else { display input string beep } S 4 : Aβ → S 4 if strlen > 0 && cunentPos < 70 { truncate cunentPos in input string and display input string } else { display input string beep } S 4 : A 7 → S 4 if strlen < 70 && cunentPos < 70 { CunentPos + + } else { display input string beep } S 4 : A 8 → S 4 if strlen < 70 && cunentPos < 70 { CunentPos- } else { display input string beep } S 4 : A 9 → S, deactivate scroll mode Additional functions Kev stroke repetition This function is activated in the states Sj and S 4 . If any of the events A 2 , A 3 , A 4 or A 5 is active longer then n seconds (where n is an integer that can be easily redefined), the event shall automatically be repeated, i.e., if the key characters A, C, G or T are pressed longer then n seconds, the character pressed will be repeatedly add to the input string as long as the key is pressed. Beep/Summer In case input, delete, scroll overrides the input string maximum length or position becomes lesser than 0 (se state definition). Although the invention has been described with regard to its prefened embodiments, which constitute the best mode presently known to the inventors, it should be understood that various changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention which is set forth in the claims appended hereto.

Claims: Claims 1. A handheld arrangement for the analysis of nucleotide sequences, characterized in that said anangement comprises: input means (1, 9) specifically adapted for entering a nucleotide sequence conesponding to the four nucleotides A, C, G and T/U, thus minimizing possible sources of enor by effecting a wrong input means; memory means (4) for storing the entered information; means (2) for analyzing the entered information; means (3, 7) for graphically displaying the secondary structure of an input sequence; and whereby said anangement is available at every work space in a laboratory environment by carrying it, thus further further minimizing the risks of introducing sources of enor. 2. An anangement according to claim 1, characterized in that it further comprises means (5) for transfe ing information wireless between said anangement and other devices. 3. An anangement according to any one of claims 1 and 2, characterized in that it comprises a timer. 4. A method for the analysis of nucleotide sequences comprising the use of a handheld anangement, characterized in that it comprises: inputing a nucleotide sequence in said anangement through input means conesponding to the four nucleotides A, C, G and T/U, thus minimizing possible sources of enor by effecting a wrong input means; storing sequence in said anangement; analyzing said sequence through said anangement; graphically displaying a secondary structure of an input sequence through said anangement; and whereby said anangement is available at every work space in a laboratory environment by carrying it, thus further minimizing the risks of introducing sources of enor. 5. A method according to claim 4, characterized by transfening information wireless between said anangement and other devices. 6. An anangement according 4 or 5, characterized in that the anangement uses a timer.

Inventors: OLSSON HELENE, OLSSON JAN, OLSSON, HELENE, OLSSON, JAN
Applicants: CYBERGENE AB, OLSSON HELENE, OLSSON JAN, OLSSON, HELENE, OLSSON, JAN
Application year: 1998
Main classification: G06F17
IPC codes: C12N15, G06F17, G06F19, G06Q50