Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-02-26
2001-01-30
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S091500, C536S023100, C536S023600, C536S024300, C536S024330
Reexamination Certificate
active
06180345
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to an improved process for simultaneous preparation of sex specific and gender-neutral semisynthetic amplicons useful for sex determination.
This invention particularly relates to an improved process for simultaneous preparation of sex specific and gender-neutral semisynthetic amplicons useful for sex determination. The invention particularly relates to a process for simultaneous preparation of sex specific and gender-neutral semisynthetic amplicons useful for sex determination of papaya plants. The process involves amplification of nucleic acid in a Polymerase Chain Reaction (PCR) using synthetic oligonucleotide primers.
BACKGROUND
Carica papaya commonly known as papaya, is a native of tropical America. It belongs to the genus Carica, a member of family caricaceae. The plant adopts itself to diverse soil and climatic conditions, and hence is grown extensively in tropical and subtropical areas for its profitable and continuous yield of fruits. The fruits are mostly consumed ripe and are a rich source of vitamins A, B, C, and D (Kumar et al.
J. Born Natural His. Society,
Vol XV, No. 2. Dec. 43, 252-256). Thin, milky latex is extracted out of young green fruits. This latex contains a high percentage of papain, which is one of the most valuable of plant proteolytic enzymes. Papain is of interest to many industries among which food industry is one of the biggest consumers. Papain is used for chillproofing beer, tenderizing meat and freeing food proteins. It is used in peptone preparation from meat and milk in bacteriological laboratories, in textile industry for degumming of silk, in dairy industry for cheese preparation, in tanning industry for skin dehairing and bating of hides, in pharmaceutical industry, perfume industry and in effluent treatment. Papain is also used to produce animal feed products and recover animal wastes by digestion of proteins. (Jones et al, Process Biochemistry 9, 1976, 21-24).
Papaya plants are propagated through seeds. The seeds are sown in seedbeds and 1-2 months old seedlings are transplanted to the field. 2-3 seedlings are planted in one pit, as the sex of the seedlings is unknown. Plants attain reproductive maturity after 5-8 months. Most of the male plants are then uprooted from the field. This unnecessary cultivation and uprooting leads to wastage of time, money and labor.
The dioecious cultivars are preferred for the extraction of papain as the female yields of crude papain exceed that of hermaphrodite and also the proteolytic activity of the crude papain from female fruits is greater than hermaphrodites (Madrigal et al., J. Sci. Food Agri. 1980, 31: 279-285).
Breeding programs are initiated with objectives to evolve disease resistant and true breeding papaya varieties with good quality fruits and high papain content. The dioecious nature poses problems and inconvenience to papaya breeders and growers since it takes 5-8 months to known the sex of the seeding. Unfortunately, sex cannot be deduced from external morphology or cytology with embryonic or juvenile forms. If the sex of plants is known at the juvenile stage, it would facilitate screening of the seedlings for female plants saving time and economic resources and thereby helping in the breeding program.
In our unpublished copending Indian Patent Application No. 268/Del/97, a process for preparation of duplex polynucleotide useful for sex determination of papaya plant has been reported. In our other unpublished copending Indian Patent Application No. 2447/Del/97 which corresponds to U.S. Ser. No. 09/052,505 filed on Mar. 31, 1998, now U.S. Pat. No. 6,037,128, a process for preparation of semisynthetic amplicon useful for sex determination of the papaya plant has been described. However, the present application more accurate and an improved process for preparation of sex specific and gender-neutral amplicons useful for sex determination. Apart from patent applications, there is no prior art available identification of sex of dioecious papaya plant describes still simultaneous semisynthetic our own-filed patent applications, there is no prior art available for the identification of sex of dioecious papaya plant.
SUMMARY
The objective of the present invention is to provide an improved process for simultaneous preparation of sex specific and gender-neutral semisynthetic amplicons useful for sex determination.
Another objective of the present invention is to provide a process for simultaneous preparation of sex specific and gender-neutral semisynthetic amplicons useful for sex determination of papaya plants.
Yet another objective of the present invention is to provide a process for simultaneous preparation of sex specific and gender-neutral semisynthetic amplicons useful for sex determination of papaya plants at juvenile stage.
DETAILED DESCRIPTION OF INVENTION
Accordingly, the present invention provides an improved process for simultaneous preparation of sex specific and gender-neutral semisynthetic amplicons useful for sex determination, which comprises, isolating nucleic acids from any part of a papaya plant by conventional methods, amplifying the said nucleic acids in a conventional Random Amplification of polymorphic DNA polymerase Chain Reaction (RAPD-PCR), resolving the amplified products by conventional electrophoresis method, eluting the sex specific, double stranded amplified product from the gel piece by known methods, cloning the said product in a known vector by conventional methods, sequencing the said cloned product by known methods, synthesizing the single stranded chains of synthetic oligonucleotides by known method based on the said sequence data, amplifying the said nucleic acid in a conventional Sequence Tagged Site Polymerase Chain Reaction by using synthetic oligonucleotides as primers to get sex specific and gender-neutral semisynthetic amplicons simultaneously.
In an embodiment of the present invention, isolation of nucleic acid can be carried out by conventional method such as Roger & Bendich method from any part of a papaya plant such as leaf, root, petiole. In an embodiment of the present invention, the polymerase chain reaction is carried out by using a thermostable DNA polymerase enzyme.
In another embodiment of the present invention, the Random Amplification of polymorphic DNA Polymerase Chain Reaction (RAPD-PCR) may be effected by using one or more stranded oligonucleotide primers, such as 5-12 mer primers, preferably known decamers.
In another embodiment of the present invention separation of the products of Polymerase Chain Reaction may be effected by conventional electrophoresis method using agarose gel, polyacrylamide gel and mixtures thereof.
In an embodiment of the present invention, elution of the sex specific amplification product from the gel piece may be effected by conventional elution methods, such as electroelution, freeze-thaw method.
In an embodiment of the present invention, cloning of the sex specific amplified, eluted product may be effected by using a plasmid vector, such as PCR product cloning vector.
In a feature of the present invention sequencing of the cloned product is effected by known methods, such as Sanger's chain termination method.
In another embodiment of the present invention the cloned semisynthetic amplicon has the following sequence.
GAGGATCCCT ATTAGTGTAA GGGATGTTCA AGAACCTAGC TCTGATATCA
50
CCTATGACAT CTCGGTACCG AATAGGGCAA CGGTGTCTGA CAACATAATA
100
GATATGAGTG CATAAAAGAA CTATACAACA GAAGAAAAGT CATTTCTTAT
150
AAAAATTTGA TGTTTAAATA CATTTGAGAT CAAGAACTTG GTAGTTAAAA
200
TATATAACAAGCATTATTATA TCAACTTCTA TATTACAAAA TAATTGTTTA
250
TCAGAGTACA ATAATTCACA TGCACTTAAA TTACGCTACA AGTTCACGAA
300
CAAATCCAAA CAAACTTTAA TGGTGCAGTT TGAGCAGCAG CAATCTTCAC
350
TTTCGTATCT CTAGGGGAAA TAGAGTTGGG GTGACTTTCA TAAGACTCAG
400
TAAACTCTGT ACGGAAAATA GTATTTAAAA TACGGTAATA AAGGTTTAAA
450
GGTTGTTTAT TTTAAAAATG TGTCATACCT TTTCATTCAA TAGAGCTTAC
500
CGTCAGAGTC CGTTGCAGAT TAAATTCATT TAAAATACTA CTAAAAAGTT
550
CATACTTTTG GTTAATTGAA ATACATTTTA AAATACCAAA ATTTCAAACA
600
TAAGCAGTAA AACTGAATG
Gupta Vidya S.
Parasnis Anjali S.
Ranjekar Prabhakar K.
Counsel of Scientific & Industrial Research
Fish & Richardson P.C.
Johannsen Diana
Myers Carla J.
LandOfFree
Process for simultaneous preparation of sex specific and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Process for simultaneous preparation of sex specific and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Process for simultaneous preparation of sex specific and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2520617