Document:

EX-10.36

                       LETTER OF INTENT REGARDING GRANT OF

                    LIMITED LICENSE IN THE REPUBLIC OF MEXICO

THIS LETTER OF INTENT, ("Letter") is entered into this 10th day of October, 2000
by and between MetaMorphix, Inc., a Delaware corporation with a business address
at 1450 South Rolling Road, Baltimore, Maryland 21227 ("MMI") and Avicola
Pilgrim's Pride de Mexico S.A. de C.V., a Mexico entity with its principal place
of business at Col San Pablo, Ave 5 De Febrero 1408, Queretaro, Queretaro,
Mexico, 76130 ("Pilgrim's Pride").

        WHEREAS, MMI is in the business of producing biotechnology-based
products focused on improving poultry production, livestock production and
animal health; and

        WHEREAS, MMI has developed Myostatin(TM) Blockers, biotechnology-based
products that inhibit the biological function of the Myostatin(TM) protein
thereby causing an increase in muscle mass and carcass weight, accelerated
weight gain, improved feeding efficiency, standardization and uniformity of
growth and/or improved nutritional profile of the meat ("Myostatin(TM)
Blockers"); and

        WHEREAS, MMI represents that it either owns or holds valid exclusive
licenses to patent applications, patents and know-how (hereinafter collectively
referred to as "Myostatin(TM) Blocker Technology") in the Republic of Mexico
related to the Myostatin(TM) Blockers for nonhuman applications that (x) either
disrupt or eliminate Myostatin(TM) protein synthesis, bind to Myostatin(TM)
receptors or interfere with Myostatin(TM) signal transduction; and (y) result in
an increase or modification of muscle growth in poultry (the "MBT
Applications"); and
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        WHEREAS, Pilgrim's Pride is in the business of producing, processing and
marketing poultry products in the Republic of Mexico; and

        WHEREAS, MMI and Pilgrim's Pride desire to enter into a long-term,
non-exclusive, non-divisible, personal license agreement with respect to the
Myostatin(TM) Blocker Technology for use by Pilgrim's Pride in poultry
production in the Republic of Mexico; and

        WHEREAS, MMI and Pilgrim's Pride desire to enter into the such license
agreement, a Confidentiality and Non-Disclosure Agreement and a Material
Transfer Agreement (collectively, the "Agreements") for the territory of the
Republic of Mexico; and

        WHEREAS, as further inducement to enter into the Agreements, MMI desires
to enter into similar agreements with Pilgrim's Pride granting a long-term,
non-exclusive, non-divisible, personal license agreement with respect to the
Myostatin(TM) Blocker Technology for use by Pilgrim's Pride in poultry
production for the territory of the Republic of Mexico (the "Limited License")

        NOW, THEREFORE, in consideration of the mutual promises and benefits set
forth herein, the adequacy of which is hereby acknowledged, the parties hereto
contemplate entering into agreements relating to the grant of the Limited
License that will reflect the following understandings:

1.      RELATIONSHIP MANAGEMENT. It is anticipated that MMI and Pilgrim's Pride
        will formulate a committee of managers which will develop protocols for
        the commercial-scale production and sale of products based on the
        Myostatin(TM) Blocker Technology in the country of Mexico (the "Mexican
        Regulatory Approval"). Such protocols will include:

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<PAGE>

        (a)     Manufacturing practices that will meet or exceed the standards
                required to obtain and maintain Mexican Regulatory Approval;

        (b)     MMI, in an effort to expedite the Mexican Regulatory Approval
                process, will make a good faith effort to seek agreement by the
                Mexican regulatory authorities to observe or, alternatively, to
                be a part of the Commercial Scale Trials referenced in the
                LOI/Limited License/Option Agreement that are required to obtain
                U.S. regulatory approval.

        (c)     Pilgrim's Pride will participate in regularly scheduled
                quarterly meetings in an effort to identify any further
                requisite scope of work that must be performed to obtain and
                maintain Mexican Regulatory Approval and to analyze the
                performance and results of any required internal research and
                trials.

2.      GRANT OF LICENSE. It is anticipated that MMI will grant the Limited
        License to Pilgrim's Pride for the limited purposes of: (1) performing
        any required research and trials to be conducted in their effort to
        obtain Mexican Regulatory Approval and (2) for use of the Myostatin(TM)
        Blocker technology limited to improving chicken meat production
        economies by inhibiting the biological function of the Myostatin protein
        by injecting Myostatin(TM) Blocker into chicken eggs. The parties
        further anticipate that:

        (a)     The territory of the Limited License will solely include the
                Republic of Mexico, without any rights of export (the
                "Territory").

        (b)     Pilgrim's Pride will execute, simultaneously with the Limited
                License, a Confidentiality and Non-Disclosure Agreement and a
                Material Transfer Agreement.

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<PAGE>

        (c)     The Limited License will bear an initial term of five years.
                Such initial term will begin upon obtaining Mexican Regulatory
                Approval, with an option to renew the Limited License for
                similar five year terms until the expiration of the license
                patents; PROVIDED, HOWEVER, that renewal of the Limited License
                will be conditioned upon Pilgrim's Pride achieving certain
                minimum sale volumes which will be mutually agreed upon by
                Pilgrim's Pride and MMI.

        (d)     MMI will waive all up-front license fees; PROVIDED HOWEVER, MMI
                and Pilgrim's Pride shall negotiate a mutually acceptable value
                added royalty payment to be paid by Pilgrim's Pride to MMI prior
                to the execution of the Limited License. The methodology will be
                based on the usual business practices of Mexico. The terms of
                the value added royalty will be similar to those terms which
                have been or will be negotiated with all other licensees of the
                territory of Mexico. Such terms shall be in compliance with all
                regulations and laws of the Republic of Mexico.

        (e)     If the parties enter into a Limited License, Pilgrim's Pride
                will provide sales and royalty reports to MMI within thirty (30)
                days after the end of each fiscal quarter.

        (f)     The Limited License will contain representations, warranties,
                covenants and indemnities by both parties customary for
                transactions and relationships of this nature.

        (g)     The Limited License shall contain provisions that (i) Pilgrim' s
                Pride may terminate the Limited License at any time by thirty
                (30) days written notice to MMI; (ii) if Pilgrim's Pride
                terminates the Limited License, Pilgrim's Pride's license to use
                the Myostatin(TM) Blocker Technology will be immediately
                revoked; (iii) MMI shall have the right to terminate the Limited
                License in the event of a

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<PAGE>

                breach by Pilgrims' Pride of any of the representations,
                warranties, covenants terms or conditions contained in the
                Limited License Agreement and where such breach has not been
                cured by Pilgrim's Pride within forty-five (45) days of receipt
                of written notice; and (iv) in the event Pilgrim's Pride fails
                to execute its Long Term License Agreement or subsequently
                terminates same with MMI for the territory of the United States,
                MMI shall have the option to either terminate the Mexico Limited
                License immediately or negotiate a mutually agreeable annual
                license fee for the territory of Mexico.

        (h)     The Limited License will not be assignable, transferable or
                saleable without MMI's prior written consent, which consent may
                be granted or withheld in MMI's sole discretion.

        (i)     Any and all discoveries related to the patent applications,
                patents or know-how arising from any required Pilgrim's Pride's
                research and trials shall be: (a) disclosed immediately to MMI;
                and (b) made available for use by MMI through a royalty-free,
                exclusive, world-wide license to the patent applications,
                patents or improvements thereof, patent rights and know-how,
                with additional rights to sublicense the license to additional
                third parties.

        (j)     Any and all discoveries related to the patent applications,
                patents or improvements thereof, patent rights and know-how
                arising after obtaining Mexican Regulatory Approval shall be:
                (a) disclosed immediately to MMI; and (b) made available for use
                by MMI through a warranty-free, nonexclusive, worldwide license
                with additional rights to sublicense the license to additional
                third parties.

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<PAGE>

        (k)     Pilgrim's Pride will agree that its internal research and trials
                of the Myostatin(TM) Blocker Technology will comply with any and
                all rules, regulations, orders, regulatory laws or restrictions
                required to obtain and maintain Mexican Regulatory Approval.

        (1)     Pilgrim's Pride will make any such data related to their
                internal research and trials available to MMI for its internal
                use and evaluation.

        (m)     Nothing in the Limited License shall be construed as preventing
                or prohibiting MMI from entering into additional agreements with
                third parties for similar activities and/or services in the
                Territory. MMI will limit the negotiation of the rights to a
                similar Limited License in the Territory to a mutually agreeable
                third party that has a substantial market share in the
                Territory, provided, however, that such limitation of
                participants will comply with all antitrust laws and regulations
                of the Republic of Mexico. Moreover, if in the event the initial
                company fails to execute a long-term license agreement or in the
                event of any governmental intervention, MMI reserves the
                exclusive right to negotiate with a replacement company or
                companies of MMI's exclusive choosing.

3.      SUPPLY OF MYOSTATIN(TM) BLOCKER PRODUCTS.

        (a)     Upon execution of the Limited License, the Confidentiality and
                Non-Disclosure Agreement and the Material Transfer Agreement,
                MMI, in its sole discretion, will supply sufficient quantities
                of the Myostatin(TM) Blocker product to Pilgrim's Pride. It is
                anticipated that MMI will be responsible for manufacturing and
                supplying the Myostatin(TM) Blocker product to Pilgrim's Pride
                to perform any required research and trials to obtain Mexican
                Regulatory Approval; PROVIDED

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<PAGE>

                HOWEVER, that the provision of such product by MMI to Pilgrim's
                Pride shall be conditioned upon such product being available in
                sufficient quantities to honor all of MMI's current commitments
                under all of MMI's existing license agreements for the United
                States.

        (b)     It is anticipated that, upon Mexican regulatory approval, MMI
                will supply, in its sole discretion, sufficient quantities of
                the Myostatin(TM) Blocker product for the use in poultry
                production in the Territory.

        (c)     Pilgrim's Pride agrees to accept such quantities of
                Myostatin(TM) Blocker product from MMI on a cost plus 10% basis.

4.      COST AND EXPENSES. The shared cost of any research and trials to obtain
        any Mexican Regulatory Approvals will be limited to $100,000 and in the
        event the cost of such research and trials exceeds $100,000, MMI will
        not seek any further contributions from Pilgrim's Pride. However, each
        party will bear separately all other individual costs and expenses in
        connection with the transactions contemplated in this Letter.

5.      INDEMNIFICATION.

        (a)     It is anticipated that Pilgrim's Pride, its directors, officers,
                employees, agents and assigns will agree to indemnify, defend
                and hold harmless MMI and its directors, officers, employees and
                agents from and against any and all claims threatened or
                initiated against MMI that arise under any Mexican or United
                States antitrust or unfair competition law, and which challenge
                any conduct contemplated herein, including, but not limited to
                the provisions of Paragraph 2(m), INFRA, or its

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<PAGE>

                implementing provision in the Limited License. This agreement to
                indemnify, defend and hold harmless shall cover any and all
                losses, expenses, damages, liabilities, and costs, including
                without limitation, interest, penalties, double or treble
                damages, reasonable attorneys' fees, court or litigation costs,
                costs associated with responding to any investigative demand or
                discovery request, judgments, and amounts paid in settlement by
                MMI (upon consultation with Pilgrim's Pride) arising from such
                claims or any claims supplemental to them. This is conditional,
                however, upon a finding and or judgment of a court of competent
                jurisdiction in the Republic of Mexico or the United States that
                the provisions contemplated herein, or its implementing
                provision in the Limited License, are unlawful, or if MME, after
                consultation with Pilgrims Pride, enters into an agreement
                settling any such actual or threatened claims.

        (b)     Each Party hereto and its directors, officers, employees, agents
                and assigns agrees to indemnify, defend and hold harmless the
                other Party hereto and its directors, officers, employees and
                agents from and against all claims, losses, damages, costs,
                expenses (including, but not limited to, reasonable attorney's
                fees and costs of litigation actually incurred) or liability
                incurred or suffered due to either of such Parties, or such
                Parties authorized agents or contractors, negligence or willful
                misconduct.

        (c)     MMI, its directors, officers, employees, agents and assigns will
                agree to indemnify, defend and hold harmless Pilgrim's Pride and
                its directors, officers,

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<PAGE>

                employees and agents from and against any and all claims related
                to Patent infringements by MMI under or related to the Limited
                License.

        (d)     The foregoing agreements to indemnify, defend and hold harmless
                shall be effective throughout the term of the Limited License,
                and shall extend to any claim initiated or threatened after the
                termination of the Limited License, if such claim arises out of
                conduct that occurred during the term of the agreement.

6.      CONFIDENTIALITY. The Confidentiality and Non-Disclosure Agreement and
        Material Transfer Agreement shall survive any termination or expiration
        of this Letter.

7.      PUBLICITY. MMI intends to issue press releases directly related to the
        execution of this Letter; PROVIDED HOWEVER, that prior to making any
        such release or discussing the transactions contemplated herein with any
        third party, with the exception of that party's legal counsel, the
        disclosing party must obtain the prior written consent of the
        nondisclosing party.

8.      RELATIONSHIP OF THE PARTIES. Nothing in this Letter shall be construed
        as preventing or prohibiting either arty from entering into agreements
        with any third parties for other activities, goods and/or services.

9.      CLOSING DATE. The parties shall negotiate in good faith towards
        execution of the various agreements containing the terms and conditions
        for the transactions contemplated herein no later than three (3) months
        from the execution of this Letter (the "Closing Date"). Such Closing
        Date will only be extended upon the mutual written consent of the
        parties.

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<PAGE>

10.     SUPERSEDED LETTER. This Letter supersedes and replaces any oral or
        written agreements between the parties that exist with reference to the
        Limited License.

        Except for provisions in this Letter relating to Cost and Expenses and
Closing Date, this Letter represents only our good faith intention to negotiate
toward execution of the Limited License and the various agreements related
thereto. This Letter is not a binding agreement between the parties and neither
party shall have any liability to the other party if either party fails to
execute the Limited License or any of the agreements related thereto for any
reason.

        Except for those Sections described herein, upon execution and delivery
of the Limited License, this Letter shall be superseded thereby, and the Limited
License, and the various agreements related thereto, shall thereafter govern the
rights and obligations of the parties with respect to the proposed transactions.

        If you agree that the foregoing correctly expresses your intent, please
sign below in the space provided. The parties shall thereafter negotiate with
each other in a positive and constructive manner with the objective of executing
a mutually satisfactory and definitive Limited License.

        IN WITNESS WHEREOF, the parties have agreed to and executed this Letter
as of the date first above written.

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<PAGE>

MMI                                     PILGRIM'S PRIDE
METAMORPHIX, INC.                       AVICOLA PILGRIM'S PRIDE DE
                                        MEXICO S.A. DE C.V.

By:/s/ Edwin C. Quattlebaum             By: /s/ David Van Hoose
   -----------------------------            -------------------------------
Name: Edwin C. Quattlebaum              Name: David Van Hoose
      --------------------                   ------------------------------
Title:Chairman, President, CEO          Title: President, CEO, COO
      --------------------------------        -----------------------------
Date of Execution:     11/15/00         Date of Execution:     11/10/00
                   -------------------                     ----------------

                                       11EX-10.37

CONFIDENTIAL  TREATMENT  REQUESTED:  Certain portions of this document have been
omitted pursuant to a request for confidential  treatment and, where applicable,
have been marked with an asterisk ("[****]") to denote where omissions have been
made. The  confidential  material has been filed  separately with the Securities
and Exchange Commission.

                                LICENSE AGREEMENT
                    MARSHFIELD CLINIC / LINKAGE GENETICS INC.

        This  License  Agreement  is made by and between  MARSHFIELD  CLINIC,  a
nonprofit tax exempt  corporation  organized and existing  under the laws of the
State of  Wisconsin  having  its  principal  office at 1000  North  Oak  Avenue,
Marshfield,  Wisconsin 54449-5777  ("MARSHFIELD"),  and LINKAGE GENETICS INC., a
Utah  corporation,  having its principal  office at 1515 W 2200 S, Suite C, Salt
Lake City, Utah 84119-1484  ("LICENSEE"),  hereafter collectively referred to as
the "PARTIES."

                                    RECITALS

        A.      MARSHFIELD is the owner by assignment of the entire right, title
and interest of certain Patent Rights (as defined below). The Patent Rights were
developed by MARSHFIELD prior to any business  relationship  between  MARSHFIELD
and LICENSEE;

        B.      LICENSEE is a biotechnical research and development firm;

        C.      MARSHFIELD  wishes to grant to LICENSEE a non-exclusive  license
to use certain Licensed  Processes to perform Licensed  Services in the Field of
Use within the Territory (as defined below), and LICENSEE wishes to receive such
a license on the terms and subject to the conditions set forth herein.

                                 1. DEFINITIONS

        1.1     "PATENT  RIGHTS" shall mean U. S. Patent Number  5,075,217  (the
'217 Patent),  entitled "Length  Polymorphisms in (dC-dA)no(dG-dT)n, Sequences,"
issued on  December  24,  1991 in the name of James L. Weber (a copy of which is
attached hereto as Attachment 1).

        1.2     "FIELD OF USE"  shall  mean  Clinical  Activities  and  Research
Activities.

        1.3     "LICENSED   PROCESSES"   shall  mean  any  IN  VITRO  diagnostic
processes the  manufacture or use of which is covered in whole or in part by the
Patent  Rights.  The  Licensed  Processes  shall  include  uses by  LICENSEE  of
inventions  covered by the Patent  Rights for Research  Activities  and clinical
activities.

        1.4     "LICENSED  SERVICES"  shall mean the performance of the Licensed
Processes by LICENSEE.

        1.5     "EFFECTIVE  DATE" shall mean January  1,1996,  regardless of the
date the Parties actually execute the Agreement.
<PAGE>

        1.6     "TERRITORY"  shall mean the United  States and its  possessions,
and the Commonwealth of Puerto Rico.

        1.7     "THIRD  PARTY"  shall  mean  a  party  other  than  LICENSEE  or
MARSHFIELD.

        1.8     "PCR   TECHNOLOGY"   shall  mean   polymerase   chain   reaction
technology,  a gene  amplification  process  currently  covered by United States
Patent Nos. 4,683,195 and 4,683,202 and other possible continuations, divisions,
reissues, and reexaminations of these patents and other patents.

        1.9     "CLINICAL  ACTIVITIES"  shall mean activities  under  LICENSEE'S
control  which are  directly  or  indirectly  related to  patient or  commercial
diagnostic or treatment purposes, or any other activity for which a fee or other
consideration  is  directly  or  indirectly  charged to a customer or patient or
received by LICENSEE.

        1.10    "RESEARCH  ACTIVITIES"  shall mean scientific  research programs
directly under LICENSEE's  control which are  specifically  directed to research
protocol and which are not clinical activities.

        1.11    "CONTRACT   YEAR"  shall  mean  the  twelve  (12)  month  period
beginning on the Effective  Date of this  Agreement,  and each twelve (12) month
period thereafter.

        1.12    "LICENSEE" shall include any wholly-owned subsidiary of LICENSEE
whose stock or other  ownership  interests is owned 100% by  LICENSEE.  The term
"LICENSEE"  shall also include its Affiliates.  An "AFFILIATE" of LICENSEE shall
mean an organization  controlled by,  controlling,  or under common control with
LICENSEE.  For this  purpose,  "control"  means  direct or  indirect  beneficial
ownership  of at least thirty  percent  (30%) of the voting  stock,  or at least
thirty  percent  (30%)  interest  in the  income  of such  corporation  or other
business, or an organization in which the amount of ownership is less than fifty
percent (50 %) and that amount is the maximum amount  permitted  pursuant to the
relevant law governing the ownership of the organization.

                          2. OWNERSHIP OF PATENT RIGHTS

        2.1     MARSHFIELD  warrants and represents  that: (i) it is a non-stock
corporation  duly organized and validly  existing under the laws of the State of
Wisconsin; (ii) the execution,  delivery, and performance of this Agreement have
been duly  authorized by all  necessary  action on the part of  MARSHFIELD;  and
(iii) it is the sole and exclusive owner of all right, title and interest in the
Patent Rights.
<PAGE>

                        3. REPRESENTATIONS AND WARRANTIES

        3.1     MARSHFIELD   warrants  and  represents   that  to  the  best  of
MARSHFIELD'S knowledge:  (i) the Patent  Rights are free and  clear of any lien,
encumbrance,  security  interest or restriction  which would  interfere with the
license  granted  under this  Agreement;  and (ii) there are no actions,  suits,
investigations,  claims or proceedings pending or threatened in any way relating
to the Patent  Rights,  and that there are no  threats  of  infringement  and no
infringement suits pending with regard to the Patent Rights.

        3.2     EXCEPT AS OTHERWISE  STATED IN THIS AGREEMENT,  MARSHFIELD MAKES
NO  REPRESENTATIONS OR WARRANTIES OF ANY KIND WITH RESPECT TO THE PATENT RIGHTS,
LICENSED  PROCESSES OR LICENSED  SERVICES  EITHER EXPRESS OR IMPLIED,  INCLUDING
WITHOUT  LIMITATION,   WARRANTIES  OF  VALIDITY,   ENFORCEABILITY,   SCOPE,  AND
MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE.

        3.3     Except as otherwise  stated in this Agreement,  MARSHFIELD makes
no representations  or warranties that the Patent Rights,  Licensed Processes or
Licensed  Services shall be free from  infringement of any patent or proprietary
rights of a third party.

        3.4     MARSHFIELD  makes no  representations  or  warranties  as to the
validity  or scope of any Patent  Rights  currently  existing or existing in the
future with respect to the Licensed Processes or Licensed Services.

                                    4. GRANT

        4.1     Subject  to the terms of this  Agreement,  MARSHFIELD  grants to
LICENSEE  and  LICENSEE  accepts a  non-exclusive  right and  license to use the
Licensed  Processes to perform Licensed  Services in the Field of Use within the
Territory.  The term of this Agreement shall begin on the Effective Date of this
Agreement  and  continue  until this  Agreement  is  terminated  as set forth in
Article 7 below.

        4.2     The Licensed Processes may be used solely for the performance of
Licensed  Services  in the Field of Use  within the  Territory  and for no other
purpose  whatsoever,  and  no  other  right,  immunity  or  license  is  granted
expressly, impliedly or by estoppel.

        4.3     LICENSEE  expressly  acknowledges  and agrees  that the  license
granted hereunder is personal to LICENSEE alone and LICENSEE shall have no right
to  sublicense,  assign or  otherwise  transfer  or share its  rights  except as
specifically  provided in this Agreement.  LICENSEE further agrees that Licensed
Services will be performed only by LICENSEE and LICENSEE shall not authorize any
other party to practice the Licensed Services.
<PAGE>

        4.4     LICENSEE  acknowledges  and  agrees  that  the  license  granted
hereunder is for the  performance  of Licensed  Services  only and  specifically
excludes any right to make,  have made,  offer or sell any  products,  including
devices,  reagents,  kits  or  diagnostic  products  for  use  in  the  Licensed
Processes.  LICENSEE  further  acknowledges and agrees that MARSHFIELD is in the
business of providing laboratory testing services and therefore may compete with
LICENSEE'S business.

        4.5     LICENSEE understands that practice of the Patent Rights requires
the use of PCR  Technology  as  defined  in  Section  1. 8 above and a  separate
license may be required to use such PCR Technology.  MARSHFIELD neither owns the
PCR Technology nor has the right to license the PCR Technology.  Therefore,  the
license granted by this Agreement is NOT to be construed:

                (a)     As a license to use the PCR Technology;

                (b)     To include licenses to contributorily infringe or induce
                        infringement  under  U.S.  law or a  foreign  equivalent
                        thereof of the PCR Technology; or

                (c)     As  consent  by  MARSHFIELD  to  any  act  which  may be
                        performed by LICENSEE,  except to the extent  allowed by
                        the licensed  Processes,  under rights herein granted to
                        the LICENSEE.

                                   5. PAYMENTS

        5.1     For  the  rights,  privileges  and  license  granted  hereunder,
LICENSEE agrees to pay MARSHFIELD the sum of [****] dollars  ($[****]) each year
the  Agreement  is in force.  The first  annual  license  fee  payment of [****]
dollars  ($[****])  (prorated  for the partial year) shall be paid no later than
July 1, 1996.  The second and  subsequent  annual  license fees shall be paid in
four (4) equal installments of [****] dollars ($[****]),  due on the last day of
each  calendar  quarter in the Contract  Year (March 31, June 30,  September 30,
December 31). The license fees applicable to any partial  Contract Year shall be
prorated on a daily basis.

        5.2     The payments set forth in this Agreement shall, if overdue, bear
interest  until payment at a per annum rate of four percent (4%) above the prime
rate in EFFECT at the Chase  Manhattan  Bank (N.A.) on the due date. The payment
of such interest shall not foreclose MARSHFIELD from exercising any other rights
it may have as a consequence of any delay in payment.

        5.3     All payments to MARSHFIELD  shall be made in U.S. dollars in the
requisite amount, at MARSHFIELD'S  option,  either to MARSHFIELD  directly or to
such bank account in the U.S.A. as MARSHFIELD may designate  without set off and
free and clear
<PAGE>

of any taxes, duties, levies, fees or charges,  except for withholding taxes (to
the extent applicable).

        5.4     LICENSEE  shall  make  payments  to  MARSHFIELD  in  the  manner
provided herein during the term of this Agreement.

                       6. PERFORMANCE OF LICENSED SERVICES

        6.1     The Parties agree that quality assurance is of utmost importance
in the performance of Licensed  Services.  To that end,  LICENSEE agrees that it
will comply with all applicable federal,  state and local regulations applicable
to human  diagnostic  testing and any other testing or  procedures  conducted by
LICENSEE hereunder.

                             7. TERM AND TERMINATION

        7.1     This  Agreement  shall commence as of the Effective Date of this
Agreement,  and, unless sooner  terminated as provided in this  Agreement,  will
terminate upon expiration of the '217 Patent.

        7.2     This Agreement may also be terminated  upon any of the following
events:

                (a)     If  LICENSEE  fails to make  timely  payment or does not
                        cure pursuant to the next sentence,  MARSHFIELD  may, at
                        its  option,  elect to  terminate  this  Agreement.  The
                        Agreement  shall be  terminated  thirty  (30) days after
                        receipt of written  notice from  MARSHFIELD to LICENSEE,
                        unless  LICENSEE  cures this  default by payment in full
                        within such thirty  (30) day period,  of all  delinquent
                        accounts  including any interest.  After two (2) or more
                        failures to make timely payment by LICENSEE,  MARSHFIELD
                        may terminate this Agreement, without LICENSEE retaining
                        any right to prevent the  termination  with a subsequent
                        cure, on ten. (10) days written  notice unless  LICENSEE
                        cures the defect within ten (10) days of notice.

                (b)     If  LICENSEE is in  material  default of any  obligation
                        hereunder,   MARSHFIELD   may  give  written  notice  to
                        LICENSEE of its intention to terminate  this  Agreement,
                        and this  Agreement  shall  terminate  thirty  (30) days
                        after the  receipt  of such  notice  unless  during  the
                        thirty (30) day period the default has been cured to the
                        reasonable satisfaction of MARSHFIELD.
<PAGE>

                (c)     MARSHFIELD may terminate this Agreement by giving thirty
                        (30) days written notice to LICENSEE if LICENSEE files a
                        petition of bankruptcy  or has any such  petition  filed
                        against  LICENSEE,  if  LICENSEE  goes  into  compulsory
                        liquidation if the business of LICENSEE is placed in the
                        possession of a receiver, a government,  or a government
                        agency,  or if  LICENSEE  makes  an  assignment  for the
                        benefit of creditors.

                (d)     LICENSEE may  terminate  this  Agreement  upon three (3)
                        month's prior written notice to MARSHFIELD in accordance
                        with Section 13.4.

                (e)     This  Agreement  shall  terminate upon the issuance of a
                        final,  unappealable  ruling  by a  court  of  competent
                        jurisdiction holding the '217 Patent to be invalid.

        7.3     Termination   of  this  Agreement   shall  not  terminate:   (i)
LICENSEE's  obligation  to make all  payments  which  shall  then  have  accrued
hereunder; and (ii) LICENSEE's obligation of confidentiality.

        7.4     Upon  termination  of this  Agreement  pursuant to any provision
hereof, LICENSEE shall:

                (a)     Immediately cease all use of the Licensed  Processes and
                        the Patent Rights; and

                (b)     Refrain from doing  anything  which would  indicate that
                        LICENSEE is associated or working with MARSHFIELD.

                          8. CONFIDENTIALITY-PUBLICITY

        8.1     The  termination  or expiration of this Agreement for any reason
shall not terminate LICENSEE'S obligations of confidentiality.

        8.2     Each party (the  "Receiving  Party")  agrees that any financial,
legal or business  information or any technical  information  disclosed to it by
the other (the  "Disclosing  Party") in connection  with this Agreement shall be
considered  confidential  and  proprietary  and the  Receiving  Party  shall not
disclose same to any Third Party and shall hold such  information  in confidence
for a period of five (5) years and will not use it other than as permitted under
this Agreement provided,  however, that any information;  know-how or data which
is orally disclosed to the Receiving Party shall not be considered  confidential
and proprietary unless such oral disclosure is reduced,  to writing and given to
the  Receiving  Party in  written  form  within  thirty  (30)  days  after  oral
disclosure thereof and marked  "Confidential." Such confidential and proprietary
information shall include, without limitation,
<PAGE>

marketing  and  sales  information,   commercialization  plans  and  strategies,
research and development  work plans,  and technical  information such as patent
applications,  inventions, trade secrets, systems, methods, apparatus,  designs,
tangible material, organisms and products and derivatives thereof.

        8.3     The above obligations of confidentiality shall not be applicable
to the extent:

                (a)     such  information is general public  knowledge or, after
                        disclosure   hereunder,   becomes   general   or  public
                        knowledge through no fault of the Receiving Party; or

                (b)     such  information can be shown by the Receiving Party by
                        its written  records to have been (i) in its  possession
                        prior   to   receipt   thereof   hereunder,    or   (ii)
                        independently  developed by the Receiving  Party without
                        any breach of this Agreement; or

                (c)     such information is received by the Receiving Party from
                        any Third Party for use or  disclosure  by the Receiving
                        Party  without any  obligation to the  Disclosing  Party
                        provided,  however,  that  information  received  by the
                        Receiving  Party  from any  Third  Party  funded  by the
                        Disclosing  Party or as an agent of the Disclosing Party
                        (e.g.. consultants,  subcontractors,  etc.) shall not be
                        released from confidentiality under this exception; or

                (d)     the disclosure of such information is required to comply
                        with or fulfill governmental  requirements,  submissions
                        to  governmental  bodies,  or the securing of regulatory
                        approvals.

        8.4     Each party shall, to the extent reasonably practicable, maintain
the  confidentiality  of the provisions of this Agreement and shall refrain from
making any public  announcement  or  disclosure  of the terms of this  Agreement
without the prior written consent of the other party, which consent shall not be
unreasonably withheld, except to the extent a party concludes in good faith that
such disclosure is required under applicable laws or regulations,  in which case
the other party shall be notified in advance of any disclosure.

               9. COMPLIANCE WITH APPLICABLE LAWS; INDEMNIFICATION

        9.1     LICENSEE  agrees  to  comply  with  all  governmental  laws  and
regulations  applicable  in  connection  with the use of any  rights  under this
Agreement. In particular, it is understood and acknowledged that the transfer of
certain  commodities  and  technical  data is subject to United  States laws and
regulations  controlling  the export of such  commodities  and  technical  data,
including all Export Administration Regulations of the United
<PAGE>

States  Department of Commerce.  These laws and regulations  among other things,
prohibit or require a license for the export of certain types of technical  data
to certain specified countries.

        9.2     In consideration  of the license granted herein,  LICENSEE shall
indemnify,  defend and hold MARSHFIELD harmless and indemnify MARSHFIELD against
and from any and all claims, demands,  losses, costs, damages, suits, judgments,
penalties,  expenses, and liabilities of any kind or nature whatsoever including
the  reasonable  attorneys'  fees that may be  incurred  by  MARSHFIELD  arising
directly or indirectly  out of or in connection  with the practice of the patent
rights or  performance  of the  Licensed  Services  by LICENSEE or other acts or
omissions of LICENSEE,  or the agents,  employees or independent  contractors of
LICENSEE.

                     10. PATENT INFRINGENENT AND ENFORCEMENT

        10.1    During  the term of this  Agreement,  MARSHFIELD  shall have the
right,  but shall not be  obligated,  to  prosecute  at its own expense any such
infringements  of the Patent Rights or Licensed  Services and, in furtherance of
such right,  LICENSEE hereby agrees that MARSHFIELD may join LICENSEE as a party
plaintiff in any such suit,  without expense to LICENSEE.  The total cost of any
such  infringement  action  commenced or defended solely by MARSHFIELD  shall be
borne by MARSHFIELD and  MARSHFIELD  shall keep any recovery or damages for past
infringement derived therefrom.

                             11. CONTEST OF VALIDITY

        11.1    In the event LICENSEE contests the validity of any of the Patent
Rights,  LICENSEE  shall  (until the Patent  Rights are  adjudicated  invalid or
unenforceable  in court)  continue to make  payments as if such contest were not
underway.  The payments shall continue in effect for the valid claims as long as
there  is at  least  one  patent  within  the  Patent  Rights  that has not been
adjudicated invalid or unenforceable.

                            12. MOST FAVORED LICENSEE

        12.1    MARSHFIELD agrees that if it should hereafter grant to any third
party in  competition  with  LICENSEE,  other than a wholly or  partially  owned
subsidiary or affiliate of  MARSHFIELD  or the United  States  government or any
agency,  or  division  thereof,  a  license  to use the  Licensed  Products  and
Processes  to perform the  Licensed  Services,  providing  for a more  favorable
annual  license  fee,  payment or other  cash or cash  equivalent  or  otherwise
readily   economically   measured   terms  with  the   exception   of  royalties
(collectively:  "More  Favored  Payment"  or  "MFP")  to  perform  the  Licensed
Services,  the  LICENSEE  shall be entitled to elect to amend this  Agreement to
provide for such more MFP,  subject to such other terms and  conditions  in such
other license as disclosed by MARSHFIELD under this Article.
<PAGE>

        12.2    This  Article  shall  not  apply  to any  future  license  which
provides  for  royalties  or license  fees based upon a  percentage  of sales or
revenues.

        12.3    MARSHFIELD  shall  disclose to LICENSEE the MFP contained in any
subsequent  license  granted  within  sixty (60) days of the  execution  of said
subsequent  license.  At the same time,  MARSHFIELD will also disclose any terms
and  conditions  in said  future  license  which  must also be  accepted  by the
LICENSEE if it elects the MFP. The LICENSEE must notify MARSHFIELD in writing of
its election to so amend this Agreement within sixty (60)days as provided above,
or LICENSEE shall forfeit the right in that regard.

        12.4    This   Article   shall  not  apply  to   forgiveness   for  past
infringements  to reach  settlement  of a lawsuit  or  genuine  dispute  between
MARSHFIELD and a Third Party with respect to the Licensed Patents.

                          13. MISCELLANEOUS PROVISIONS

        13.1    ASSIGNABILITY:  This  Agreement and the License  herein  granted
shall not be assignable except with the prior written consent of MARSHFIELD.

        13.2    TRADEMARK AND NAME RIGHTS:  Except as otherwise provided in this
Agreement,  no right, express or implied, is granted by this Agreement to use in
any manner the names "Marshfield Clinic,"  "Marshfield,"  "Linkage Genetics," or
any  other  trade  name  or  trademark  of  either  party  or  any   simulation,
abbreviation, or adaptation of the same, or the name of any employee or agent of
a party hereto, without the prior written express consent of such party.

        13.3    RELATIONSHIP  BETWEEN PARTIES:  MARSHFLELD and LICENSEE are both
independent  contractors  and not joint  venturers  with or partners,  agents on
employees of each other.  Neither  party shall in any respect by act or omission
represent or simply or permit any  representation  or  implication by the act or
omission of another that it is a joint venturer  with, or the partner,  agent or
employee  of the other,  or in any respect  authorized  or  empowered  to act on
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Agreement. This Agreement does not create a franchise or dealership.

        13.4    NOTICES:  All  notices  and  other  communications  to be  given
hereunder  shall be  effective  upon  receipt.  Such  notices  shall be given in
writing by  registered  or  certified  mail,  postage  prepaid,  return  receipt
requested,  or by mail-gram telex or facsimile to the respective  parties at the
following  addresses,  unless such  addresses are changed and the other Party is
notified of the change in writing:
<PAGE>

For Legal Notices:

LICENSEE:

                Linkage Genetics Inc.
                1515 W. 2200 S, Suite C
                Salt Lake City, Utah 84119-1484
                Attention:_______________________________________

MARSHFIELD:

                Marshfield Clinic
                1000 North. Oak Avenue
                Marshfield, Wisconsin 54449-5777
                Attention:     Michael G. May, Esq.

With a copy to:

                Charles S. Sara, Esq.
                DeWitt Ross & Stevens S. C.
                Firstar Financial Centre
                8000 Excelsior Drive, Suite 401
                Madison, Wisconsin 53717-1914

For Notices relating to technical or business matters:

LICENSEE:

                Linkage Genetics Inc.
                1515 W 2200 S, Suite C
                Salt Lake City, Utah 84119-1484
                Attention:_______________________________________

MARSHFIELD:

                Marshfield Clinic
                1000 North Oak Avenue
                Marshfield, Wisconsin 54449-5777
                Attention:    Michael G. May, Esq.

        13.5    ENTIRE  AGREEMENT:  The  parties  hereto  acknowledge  that this
Agreement and its Attachments set forth the entire  agreement and  understanding
of the parties hereto as to the subject  matter hereof,  and supersede all prior
discussions, agreements and writings with respect hereto.

        13.6    LAW TO GOVERN:  This Agreement shall be deemed to have been made
in the State of Wisconsin,  and shall be construed, and the respective rights of
the parties  hereunder  determined,  in accordance with the laws of the State of
Wisconsin.
<PAGE>

        13.7    SEVERABILITY:  All agreements and covenants contained herein are
severable,  and in the  event  any of them  shall be held to be  invalid  by any
competent  court,  this  contract  shall  be  interpreted  as  if  such  invalid
agreements or covenants were not contained herein.

        13.8    MODIFICATION; WAIVER: This Agreement may not be altered, amended
or modified in any way except by a writing  signed by both parties.  The failure
of a party to enforce any provision of the  Agreement  shall not be construed to
be a waiver of the right of such party to thereafter  enforce that  provision or
any other provision of that right.

        13.9    COUNTERPARTS:  This  Agreement  may be  executed  in two or more
counterparts, each of which shall be deemed an original and which together shall
constitute one instrument.

        13.10   FORCE  MAJEURE:  No party shall be liable for failure to perform
or delay in  performing  obligations  set forth in the  Agreement,  and no party
shall be deemed in breach or default of its  obligations,  if, to the extent and
for so long as,  such  failure,  delay,  breach,  or  default  is due to natural
disasters or any similar causes reasonably beyond the control of such party. Any
party desiring to invoke the  protection of Force Majeure shall promptly  notify
the  other  party of such  desire  and shall use  reasonable  efforts  to resume
performance of its obligations.

        IN WITNESS  WHEREOF,  the undersigned have executed this Agreement as of
the Effective Date set forth above.

LINKAGE GENETICS INC.

By      /s/ Scott H. Wright
    --------------------------------------------------
         Name: Scott H. Wright
         Title:  Vice President

Date

By      /s/ Richard A. Leer, M.D.
    --------------------------------------------------
    Richard A. Leer, M.D., President Date      8/8/96

Date     8/8/96
    -------------------------------------------------

<PAGE>

                                  ATTACHMENT 1

UNITED STATES PATENT                    PATENT NUMBER:                 5,075,217

WEBER                                   DATE OF PATENT:        DECEMBER 24, 1991

--------------------------------------------------------------------------------
Length polymorphisms in (dC-dA)no(dG-dT)n sequences

                                    ABSTRACT

The present invention is directed to a product and process for characterizing
the human genome utilizing polymorphic DNA fragments containing (dC-dA)n
..(dG-dT)n. The process comprises identifying polymorphic DNA fragments
containing the above tandem repeat sequence followed by characterizing
admixtures of genomic material by amplification of fragments containing the
tandem repeat sequence using substantially unique and non-polymorphic primer
sequences and appropriate separation of the so amplified fragments.

--------------------------------------------------------------------------------
Inventors:  WEBER; JAMES L. (Marshfield, WI)
Assignee:   MARSHFIELD CLINIC (Marshfield, WI)
Appl. No.:  07/341,562
Filed:      APRIL 21, 1989

--------------------------------------------------------------------------------
CURRENT U.S. CLASS:                                      435/6 ; 435/5; 536/25.5
CURRENT INTERNATIONAL CLASS:                                C12Q 1/68 (20060101)
FIELD OF SEARCH:                                          435/6 536/27 935/77,78

--------------------------------------------------------------------------------
                        REFERENCES CITED [REFERENCED BY]

--------------------------------------------------------------------------------
                              U.S. PATENT DOCUMENTS

   4582788              April 1986                  Erlich
   -------
   4623619              November 1986               Owerbach et al.
   -------
   4666828              May 1987                    Gusella
   -------
   4681840              July 1987                   Stephenson et al.
   -------
   4683194              July 1987                   Saiki et al.
   -------
   4683202              July 1987                   Mullis
   -------
   4710461              December 1987               Komano et al.
   -------
   4710465              December 1987               Weissman et al.
   -------

                            FOREIGN PATENT DOCUMENTS

    186271                         Oct., 1985                     GB
    238329                         Mar., 1987                     GB
<PAGE>

                                OTHER REFERENCES

Weber, et al., "Genome Mapping", Cold Spring Harbor Meeting, Apr. 27-May 1,
1988. .

Weber, et al., "Abundant New Class of Human DNA Polymorphisms", Am. J. Hum.
Genet. 1989 44(3), pp. 388-396. .

Aldridge, et al., "A Strategy to Reveal High-Frequency RFLPs Along the Human X
Chromosome", Am. J. Hum. Genet. 36:546-564, 1984. .

Botstein, et al., "Construction of a Genetic Linkage Map in Man Using
Restriction Fragment Length Polymorphisms", Am. J. Hum. Genet. 32:314-331,
1980. .

Braaten, et al., "Locations and Contexts of Sequences that Hybridize to Poly
(dG-dT).multidot.(dC-dA) in Mammalian Ribosomal DNAs and Two X-Linked Genes",
Nucleic Acids Research, vol. 16, #3 1988. .

Das, et al., "The Human Apolipoprotein C-II Gene Sequence Contains a Novel
Chromosome 19-Specific Minisatellite in Its Third Intron", The Journal of
Biological Chemistry 1987. .

Gilliam, et al., "Isolation of Polymorphic DNA Fragments From Human Chromosome
4", Nucleic Acids Research, vol. 15, #4, 1987. .

Gross, et al., "Chromatin Structure of the Potential Z-Forming Sequence
(dT-dG)n.(dC-dA)n ", J. Mol. Bio. (1985) 183.251-265. .

Gross, et al., "The Ubiquitous Potential Z-Forming Sequence of Eucaryotes,
(dT-dG)n.(dC-dA)n, Is Not Detectable in the Genomes of Eubacteria,
Archaebacteria, or Mitrochondria", Molecular and Cellular Biology, Aug. 1986 pp.
3010-3013. .

Hamada, et al., "Potential Z-DNA Forming Sequences are Highly Dispersed in the
Human Genome", Nature vol. 298, Jul. 22, 1982. .

Hamada, et al., "A Novel Repeated Element with Z-DNA-Forming Potential is Widely
Found in Evolutionarily Diverse Eukaryotic Genomes", Proc. Natl. Acad. Sci.
U.S.A. 79 (1982). .

Hamada, et al., "Characterization of Genomic Poly(dT-dG).Poly (dC-dA) Sequences:
Structure, Organization, and Conformation", Molecular and Cellular Biology, Dec.
1984, pp. 2610-2621. .

Hamada, et al., "Enhanced Gene Expression by the Poly (dT-dG).Poly (dC-dA),
Sequence", Molecular and Cellular Biology, Dec. 1984, pp. 2622-2630. .

Jeffreys, et al., "Spontaneous Mutation Rates to New Length Alleles at
Tandem-Repetitive Hypervariable Loci in Human DNA", Nature vol. 332, Mar. 17,
1988. .

Jeffreys, et al., "Hypervariable `Minisatellite` Regions in Human DNA", Nature,
vol. 314 Mar. 7, 1985. .

Miesfeld, et al., "A Member of a New Repeated Sequence Family Which is Conserved
Throughout Eucaryotic Evolution is Found Between the Human .delta. and .beta.
Globin Genes", Nucleic Acids Research, vol. 9, #22, 1981. .

Mullis, et al., "Specific Synthesis of DNA in Vitro via a Polymerase-Catalyzed
Chain Reaction", Methods in Enzymology, vol. 155, 1987. .

Nakamura, et al., "Variable Number of Tandem Repeat (VNTR) Markers for Human
Gene Mapping", Science, vol. 235, Mar. 27, 1987. .

Nordheim, et al., "The Sequence (dC-dA)n.(dG-dT)n Forms Left-Handed Z-DNA in
Negatively Supercoiled Plasmids", Proc. Natl. Acad. Sci. U.S.A., vol. 80, pp.
1821-1825, Apr. 1983. .

Overhauser, et al., "Identification of 28 DNA Fragments That Detect RFLPs in 13
Distinct Physical Regions of the Short Arm of Chromosome 5", Nucleic Acids
Research, vol. 15, #11, 1987.
<PAGE>

Pardue, et al., "(dC-dA)n.(dG-dT)n Sequences Have Evolutionarily Conserved
Chromosomal Locations in Drosophila with Implications for Roles in Chromosome
Structure and Function", The EMBO Journal, vol. 6, #6, pp. 1781-1789, 1987.

Saiki, et al., "Enzymatic Amplification of beta-Globin Genomic Sequences and
Restriction Site Analysis for Diagnosis of Sickle Cell Anemia", Science, vol.
20, Dec. 1985. .

Saiki, et al., "Primer-Directed Enzymatic Amplification of DNA with a
Thermostable DNA Polymerase", Science, vol. 239, Jan. 29, 1988. Schumm, et al.,
"Identification of More Than 500 RFLPs by Screening Random Genomic Clones", Am.
J. Hum. Genet. 42:143-159, 1988.

Shen, et al., "Sequence of the Human Somatostatis I Gene", Science, vol. 224,
Apr. 13, 1984. .

Skolnick, et al., "Simultaneous Analysis of Multiple Polymorphic Loci Using
Amplified Sequence Polymorphisms (ASPs)", Genomics 2, 273-279 (1988).

Slightom, et al., "Human Fetal.sup.G .gamma and .sup.A .gamma-Globin Genes:
Complete Nucleotide Sequences Suggest That DNA Can be Exchanged Between These
Duplicated Genes", Cell, vol. 21, 627-638, Oct. 1980.

Sun, et al., "Non-Alu Family Interspersed Repeats in Human DNA and Their
Transcriptional Activity", Nucleic Acids Research, vol. 12, #6, 1984.

Tautz, et al., "Simple Sequences are Ubiquitous Repetitive Components of
Eukaryotic Genomes", Nucleic Acids Research, vol. 12, #10, 1984.

PRIMARY EXAMINER: Wax; Robert A.
ASSISTANT EXAMINER: Fleisher; Mindy B.
ATTORNEY, AGENT OR FIRM: Andrus, Sceales, Starke & Sawall
--------------------------------------------------------------------------------
                                     CLAIMS

--------------------------------------------------------------------------------

I claim:

1. A method of analyzing or typing polymorphic DNA fragments which contain
(dC-dA)n.(dG-dT)n repeats which are specific to particular loci in the genome
comprising:

amplifying at least one polymorphic DNA fragment containing at least one
(dC-dA)n.(dG-dT)n segment using the polymerase chain reaction with a DNA sample
which includes the said DNA fragment as a template, and with two oligonucleotide
primers sufficiently complementary to non-repeated sequences at the ends of said
fragment within said sample to hybridize therewith, and separating and
characterizing amplified fragments on the basis of size.

2. The method of claim 1 further comprising labeling or marking said polymorphic
DNA fragments.

3. The method of claim 1 wherein said primers are obtained by direct synthesis.

4. The method of claim 1 wherein said primers are end-labeled.

5. The method of claim 1 wherein more than one DNA fragment containing at least
one segment of

<PAGE>

(dC-dA)n.(dG-dT)n is amplified and characterized simultaneously.

6. The method of claim 1 used to establish the identity, pedigree, or
relationship of an individual comprising comparison of said amplified fragments
with amplified fragments from the DNA of one or more control individuals wherein
the control DNA samples have been similarly amplified and characterized.

7. The method of claim 1 wherein the amplified fragments are separated and
characterized by gel electrophoresis.

8. The method of claim 1 wherein the DNA sample is genomic DNA.

9. The method of claim 1 wherein the DNA sample is cloned DNA. [GRAPHIC OMITTED]

--------------------------------------------------------------------------------
                                   DESCRIPTION

--------------------------------------------------------------------------------

BACKGROUND AND FIELD OF THE INVENTION

The invention relates to polynucleotides which comprise an abundant new class of
DNA polymorphisms and to a method for analyzing these polymorphisms. The
polymorphisms can be used to identify individuals such as in paternity and
forensic testing, and can also be used to map genes which are involved in
genetic diseases or in other economically important traits.

The vast majority of DNA in higher organisms is identical in sequence among
different individuals (or more accurately among the chromosomes of those
individuals). A small fraction of DNA, however, is variable or polymorphic in
sequence among individuals, with the formal definition of polymorphism being
that the most frequent variant (or allele) has a population frequency which does
not exceed 99% (Gusella, J. F. (1986), Ann. Rev. Biochem. 55:831-854). In the
past, polymorphisms were usually detected as variations in gene products or
phenotypes such as human blood types. Currently, almost all polymorphisms are
detected directly as variations in genomic DNA.

Analysis of DNA polymorphisms has relied on variations in the lengths of DNA
fragments produced by restriction enzyme digestion. Most of these restriction
fragment length polymorphisms (RFLPs) involve sequence variations in one of the
recognition sites for the specific restriction enzyme used. This type of RFLP
contains only two alleles, and hence is relatively uninformative. A second type
of RFLP is more informative and involves variable numbers of tandemly repeated
DNA sequences between the restriction enzyme sites. These polymorphisms called
minisatellites or VNTRs (for variable numbers of tandem repeats) were developed
first by Jeffreys (Jeffreys et al (1985), Nature 314:67-73).

Jeffreys has filed two European patent applications, 186,271 and 238,329 dealing
with the minisatellites. The first Jeffreys' application ('271) identified the
existence of DNA regions containing hypervariable tandem repeats of DNA.
Although the tandem repeat sequences generally varied between minisatellite
regions, Jeffreys noted that many minisatellites had repeats which contain core
regions of highly similar sequences. Jeffreys isolated or cloned, from genomic
DNA, polynucleotide probes comprised essentially of his core sequence (i.e.,
wherein the probe had at least 70% homology with one
<PAGE>

of his defined cores). These probes were found to hybridize with multiple
minisatellite regions (or loci). The probes were found to be useful in forensic
or paternity testing by the identification of unique or characteristic
minisatellite profiles. The later Jeffreys' European patent application proposed
the use of probes which were specific for individual minisatellites located at
specific loci in the genome. One problem with the Jeffrey's approach is that
some of the most highly variable and hence useful minisatellites are susceptible
to significant frequencies of random mutation (Jeffreys et al (1988), Nature
332:278-281).

Other tandemly repeated DNA families, different in sequence from the Jeffreys
minisatellites, are known to exist. In particular, (dC-dA)n(dG-dT)n sequences
have been found in all eukaryotes that have been examined. In humans there are
50,000-100,000 blocks of (dC-dA)n.(dG-dT)n sequences, with n ranging from
about 15-30 (Miesfeld et al (1981), Nucleic Acids Res. 9:5931-5947; Hamada and
Kakunaga (1982), Nature 298:396-398; Tautz and Renz (1984), Nucleic Acids Res.
12:4127-4138). Prior to the work of this invention, a number of different human
blocks of (dC-dA)n.(dG-dT)n repeats had been cloned and sequenced, mostly
unintentionally along with other sequences of interest. Several of these
characterized sequences were analyzed independently from two or more alleles. In
arriving at this invention, sequences from these different alleles were
compared; variations in the number of repeats per block of repeats were found in
several cases (Weber and May (1989), Am. J. Hum. Genet. 44:388-396)
(incorporated herein by reference in its entirety). The majority of these cases
were not noted in publications. Although three isolated research groups produced
published notations of site specific differences in sequence length (Das et al
(1987), J. Biol. Chem. 262:4787-4793; Slightom et al (1980), Cell 21:627-638;
Shen and Rutter (1984), Science 224:168-171), none of the groups recognized nor
appreciated the extent of this variability or its usefulness and none
generalized the observation. The other groups also did not consider the use of
(dC-dA)n.(dG-dT)n sequences as genetic markers and did not offer a method by
which such polymorphisms might be analyzed.

SUMMARY OF THE INVENTION

It has been discovered that (dC-dA)n.(dG-dT)n sequences exhibit length
polymorphisms and therefore serve as an abundant pool of potential genetic
markers (Weber and May (1988), Am. J. Hum. Genet. 43:A161 (abstract); Weber and
May (1989), Am. J. Hum. Genet. 44:388-396) (both incorporated herein by
reference). Accordingly, as a first feature of the present invention,
polynucleotides are provided consisting of a DNA fragment, preferably
..ltoreq.300 base pairs (bp) in length, containing one or more blocks of tandem
dinucleotide repeats (dC-dA)n.(dG-dT)n where n is preferably .gtoreq.6.

A further aspect of the invention is the provision of a method for analyzing one
or more specific (dC-dA)n.(dG-dT)n polymorphisms individually or in
combination, which involves amplification of a small segment(s) of DNA
(containing the block of repeats and some non-repeated flanking DNA) starting
with a DNA template using the polymerase chain reaction, and sizing the
resulting amplified DNA, preferably by electrophoresis on polyacrylamide gels.
In a preferred embodiment, the amplified DNA is labeled during the amplification
reaction by incorporation of radioactive nucleotides or nucleotides modified
with a non-radioactive reporter group.

A further aspect of the invention is the provision of primers for the
amplification of the polymorphic
<PAGE>

tandemly repeated fragments. The primers are cloned, genomic or preferably
synthesized and contain at least a portion of the non-repeated, non-polymorphic
flanking region sequence.

A further aspect of the invention is the provision of a method for determining
the sequence information necessary for primer production through the isolation
of DNA fragments, preferably as clones, containing the (dC-dA)n.(dG-dT)n
repeats, by hybridization of a synthetic, cloned, amplified or genomic probe,
which contains a sequence that is substantially homologous to the tandemly
repeated sequence(dC-dA)n.(dG-dT)n, to the DNA fragment. In a preferred
embodiment the probe would be labeled, e.g., end labelling, internal labeling or
nick translation.

In a more particular aspect of the present invention, some or all of the
polynucleotide primers are (32) P or (35) S labeled in any conventional manner,
such as end labeling, interior labeling, or post reaction labeling. Alternative
methods of labeling are fully within the contemplation of the invention such as
biotin labeling or possibly enzyme labeling (Matthews and Kricka (1988), Anal.
Biochem. 169:1-25).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an example of a human (dC-dA)n.(dG-dT)n polymorphism showing the
sequence of the amplified DNA, the primers used in the amplification, and an
autoradiograph of a polyacrylamide gel loaded with amplified DNA from this
marker.

FIG. 2 is an additional example of length polymorphisms in amplified fragments
containing (dC-dA)n.(dG-dT)n sequences. Shown is an autoradiograph of a
polyacrylamide gel.

FIG. 3 is an autoradiograph of a polyacrylamide gel loaded with DNA amplified
from the human Mfd3, ApoAII locus using DNA from three unrelated individuals as
template and labeled through three different approaches: labeling the interiors
of both strands with alpha (32) P-dATP, end-labeling the GT-strand primer with
(32) P phosphate, or end-labeling the CA-strand primer with (32) P phosphate.

FIG. 4 is an example of the Mendelian inheritance of three different human
(dC-dA)n.(dG-dT)n markers through three generations. Shown are the pedigree of
this family, an autoradiograph of a gel loaded with the amplified DNA and a list
of the genotypes of the individual family members.

FIG. 5 is an autoradiograph showing treatment of amplified DNA containing
(dC-dA)n.(dG-dT)n sequences with either the Klenow fragment of DNA polymerase I
or with T4 DNA polymerase.

FIG. 6 is an autoradiograph showing the effect of additional polymerase chain
reaction cycles on amplified DNA for the Mfd3 marker from a single individual.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Development of a polymorphic DNA marker based on length variations in blocks of
(dC-dA)n.(dG-dT)n repeats involves the following steps. First the sequence of a
segment of DNA containing the repeats must be determined. This is accomplished
most commonly by selecting a genomic DNA clone
<PAGE>

through hybridization to synthetic poly(dC-dA).poly(dG-dT) and then subsequently
sequencing that clone. This same step can also be accomplished simply by
selecting a suitable sequence from the literature or from one of the DNA
sequence databases such as GenBank. The later approach is severely limited by
the relatively small number of (dC-dA)n.(dG-dT)n sequences that have been
published. Second, once the sequence to be used is in hand, a pair of
appropriate primers can be synthesized which are at least partially
complementary to non-repeated, non-polymorphic sequences which flank the block
of dinucleotide repeats on either side. Third, these primer pairs are used in
conjunction with a genomic DNA (or occasionally cloned DNA) template to amplify
a small segment of DNA containing the repeats using the polymerase chain
reaction (Saiki et al (1985), Science 230:1350-1354 and U.S. Pat. No. 4,683,202,
the substance of which is incorporated herein in its entirety). The DNA is
preferably labeled during the amplification process by incorporating radioactive
nucleotides. Fourth, the amplified DNA is resolved by polyacrylamide gel
electrophoresis in order to determine the sizes of these fragments and hence the
genotypes of the genomic DNA donors.

To exemplify the informativeness of the CA-GT repeats, sequences from 25
different human polymorphic markers of the type which can be used within the
present invention, are listed in Table 1 in Example I. Each sequence represents
only one allele at each specific locus. The first five sequences were taken from
a computer search of GenBank; the remaining sequences were determined in the
laboratory (see Example I). As can be seen in this compilation, the sequences
exhibit substantial variation in the form of the tandem repeats. Some sequences,
like for example markers Mfd3, Mfd17 and Mfd23, contain only CA-GT repeats with
no imperfections. Other sequences, like for example Mfd2, Mfd7, Mfd13, and Mfd19
contain in addition to long runs of perfect CA-GT repeats, one or more
imperfections in the run of repeats. These imperfections can be additional bases
as in Mfd2 or more frequently GA-TC, AT-TA or CG-GC dinucleotide repeats as in
Mfd7, Mfd13 and Mfd19. Homogenous runs of other dinucleotide repeats are often
found in association with the CA-GT repeats like for example in Mfd5 and Mfd21.
All of these repeat sequences can be used in this application.

Every human (dC-dA)n.(dG-dT)n sequence with 11 or more repeats that has been
tested by the invention has been found to be polymorphic (over 30 sequences to
date). Since there are an estimated 50,000-100,000 (dC-dA)n.(dG-dT)n blocks in
the human genome, blocks are separated by an average spacing of 30,000-60,000 bp
which is extremely tight in genetic terms. Two polymorphic markers spaced so
that there is only 1% recombination between them are generally thought to be
about 10.sup.6 bp apart; markers spaced only 50,000 bp apart on the average
would be coinherited 99.95% of the time. This means that (dC-dA)n.(dG-dT)n
markers should find significant usage in the genetic mapping and clinical
diagnosis of human genetic diseases, much as RFLPs have been used in the mapping
and diagnosis of diseases such as cystic fibrosis (White and Lalouel (1988),
Ann. Rev. Genet. 22:259-279).

The correspondence between polymorphisms (which are relatively rare in the
genome) and the (dC-dA)n.(dG-dT)n sequences is very strong evidence that the
repeats are mainly, if not entirely, responsible for the sequence length
variations. Further evidence comes from the fact that amplified polymorphic
fragments containing the (dC-dA)n.(dG-dT)n sequences always differ in size by
multiples of 2 bp. Direct sequencing (see Example VI below) of allelic DNA also
confirms this interpretation.

The informativeness of the (dC-dA)n.(dG-dT)n polymorphisms is good to very good,
with
<PAGE>

heterozygosities ranging from 34-91%. Most of the (dC-dA)n.(dG-dT)n markers are
therefore more informative than the two-allele RFLPs (Donis-Keller et al (1987),
Cell 51:319-337; Schumm et al (1988), Am. J. Hum. Genet. 42:143-159). The number
of alleles counted for the (dC-dA)n.(dG-dT)n markers tested to date has ranged
from 4-11. Relatively high numbers of alleles also improves the usefulness of
these markers. Alleles tend to differ by relatively few numbers of repeats, with
the result that all alleles for a single marker may span a range in size of 20
bp or less. This means that amplified fragments from several different markers
can be analyzed simultaneously on the same polyacrylamide gel lanes, greatly
improving the efficiency of the amplification process and the ability to
identify individuals using the test.

In addition to the inherent useful properties of the (dC-dA)n.(dG-dT)n markers,
the use of the polymerase chain reaction to analyze the markers offers
substantial advantages over the conventional blotting and hybridization used to
type RFLPs. One of these advantages is sensitivity. Whereas microgram amounts of
DNA are generally used to type RFLPs, ten nanograms of genomic DNA is sufficient
for routine genotyping of the (dC-dA)n.(dG-dT)n block markers (FIG. 3), and the
polymerase chain reaction has recently been described as capable of amplifying
DNA from a single template molecule (Saiki et al (1988), Science 239:487-491).
Enough DNA can be isolated from a single modest blood sample to type tens of
thousands of (CA)n block markers. Another advantage of the polymerase chain
reaction is that the technique can be partially automated. For example, several
commercial heating blocks are available which can automatically complete the
temperature cycles used for the polymerase reaction. Automatic amplification
reactions and the capability to analyze hundreds of markers on each
polyacrylamide gel mean that the (dC-dA)n.(dG-dT)n markers can be analyzed
faster than RFLPs and are more readily usable in practical applications such as
identity testing.

The practical outer limits of the length of the amplified DNA fragment is
generally limited only by the resolving power of the particular separation
system employed. The thin denaturing gels used in the work leading to this
application are capable of resolving fragments differing by as little as 2 bases
up to a total fragment length of about 300 bp. Use of longer gels and longer
electrophoresis times could extend the resolving power up to perhaps 600 bp or
even more. However the longer the fragment the lower the proportion of its
length will be made up of the (dC-dA)n.(dG-dT)n sequences, and hence the more
difficult the resolution.

One specific application for the present invention is in the identification of
individual humans such as in paternity and maternity testing, immigration and
inheritance disputes, zygosity testing in twins, tests for inbreeding in man,
evaluation of the success of bone marrow transplantation, quality control of
human cultured cells, identification of human remains, and testing of semen
samples, blood stains, and other material in forensic medicine. In this
application, the ability to run numerous markers in a single amplification
reaction and gel lane gives this procedure the possibility of extreme efficiency
and high throughput.

Another specific application would be in human genetic analysis, particularly in
the mapping through linkage analysis of genetic disease genes and genes
affecting other human traits, and in the diagnosis of genetic disease through
coinheritance of the disease gene with one or more of the polymorphic
(dC-dA)n.(dG-dT)n markers.
<PAGE>

A third specific application contemplated for the present invention is in
commercial animal breeding and pedigree analysis. All mammals tested for
(dC-dA)n.(dG-dT)n sequences have been found to contain them (Gross and Garrard
(1986), Mol. Cell. Biol. 6:3010-3013). Also, as a byproduct of efforts to
develop (dC-dA)n.(dG-dT)n markers specific for human chromosome 19 from a
library developed from a hamster-human somatic cell hybrid, several hamster
(dC-dA)n.(dG-dT)n markers have been developed (Weber and May (1989), Am. J. Hum.
Genet. 44(3):388-396).

A fourth specific application is in commercial plant breeding. Traits of major
economic importance in plant crops can be identified through linkage analysis
using polymorphic DNA markers. The present invention offers an efficient new
approach to developing such markers for various plant species.

It is also contemplated that the present invention and method of
characterization could be easily extended to include other tandemly repeated
simple sequences which may be polymorphic. Examples include (dG-dA)n.(dC-dT)n,
(dT-dA)n.(dA-dT)n, and even (dA)n.(dT)n.

EXAMPLES

Example I

This example describes the method used to identify and isolate specific (dC-dA)
fragments.

General Procedure

Total human genomic DNA or total DNA from a chromosome 19-specific large insert
page library (LL19NL01) was digested to completion with Sau3A I, Alu I, Taq I,
or a combination of Sau3A I and Taq I. DNA fragments ranging in size from about
150 to 400 base pairs were purified by preparative agarose gel electrophoresis
(Weber et al (1988), J. Biol. Chem. 263:11321-11425), and ligated into mp18 or
mp19 m13 vectors. Nitrocellulose plaque lifts (Benton and Davis (1977), Science
196:180-182) prepared from the resulting clones were screened by hybridization
to synthetic poly(dC-dA).poly(dG-dT) which had been nick-translated using both
alpha (32) P-dATP and alpha (32) P-dTTP to a specific activity of about
5.times.10.sup.7 cpm/.mu.g. Hybridizations were carried out in 6XSSC, pH 7.0,
2.5 mM EDTA, 5.0% (v/v) O'Darby Irish Cream Liqueur at 65(o). After
hybridization, filters were washed in 2XSSC, 25 mM NaPO4, 0.10% SDS, 5.0 mM
EDTA, 1.5 mM Na4 P2 O7, pH 7.0, and then in 1XSSC, 0.10% SDS, 5.0 mM EDTA, pH
7.0. Phage from the first screen were usually diluted and then screened a second
time to insure plaque purity. Single stranded DNA was isolated from the positive
clones and sequenced as described (Biggin et al (1983), Proc. Natl. Acad. Sci.
USA 80:3963-3965).

GenBank DNA databases were screened for the presence of sequences with (dC-dA)6
or (dG-dT)6 using the QUEST program made available by Intelligenetics Inc.
through the national BIONET computing network. Since the sequences of only one
of the two strands of each DNA fragment are compiled in GenBank, separate
screens for both CA and GT repeats are necessary.
<PAGE>

Results

The hybridization procedure was used to isolate and sequence over one hundred
(dC-dA)n.(dG-dT)n blocks and the DNA immediately flanking the repeats. Examples
are listed in Table 1 (Mfd6-25). Numbers of CA-GT dinucleotide repeats within
the blocks ranged from 10 to over 30. Many of the blocks had imperfect repeats
or were adjacent to tandem repeats with different sequences.

(dC-dA)n.(dG-dT)n sequences obtained from the GenBank screens (Mfd1-5) were
similar to those obtained through the hybridization procedure, except that
sequences containing as few as six repeats could be selected.

TABLE 1

<TABLE>
<CAPTION>
---------------------------------------------------------------------------------------
MARKER     REPEAT SEQUENCE                  PRIMER SEQUENCES
---------------------------------------------------------------------------------------
<S>    <C>                              <C>
Mfd1   CATA(CA)19                       GCTAGCCAGCTGGTGTTATT: ACCACTCTGGGAGAAGGGTA
Mfd2   (AC)13 A(AC)17 A                 CATTAGGATGCATTCTTCTG: GTCAGGATTGAACTGGGAAC
Mfd3   (CA)16 C                         GGTCTGGAAGTACTGAGAAA: GATTCACTGCTGTGGACCCA
Mfd4   (AC)12 GCACAA(AC)13 A            GCTCAAATGTTTCTGCAACC: CTTTGTAGCTCGTGATGTGA
Mfd5   (CT)7 (CA)23                     CATAGCGAGACTCCATCTCC: GGGAGAGGGCAAAGATCGAT
Mfd6   (CA)5AA(CA)13                    TCCTACCTTAATTTCTGCCT: GCAGGTTGTTTAATTTCGGC
 Mfd7  (CA)20TA(CA)2                    GTTAGCATAATGCCCTCAAG: CGATGGAGTTTATGTTGAGA
Mfd8   (AC)20A                          CGAAAGTTCAGAGATTTGCA: ACATTAGGATTAGCTGTGGA
Mfd9   (CA)17                           GATGTCTCCTTGGTAAGTTA: AATACCTAGGAAGGGGAGGG
Mfd10  (AC)14A                          CATGCCTGGCCTTACTTGC: AGTTTGAGACCAGCCTGCG
Mfd11  (AC)23A                          ACTCATGAAGGTGACAGTTC: GTGTTGTTGACCTATTGCAT
Mfd12  (AC)11AT(AC)8A                   GGTTGAGATGCTGACATGC: CAGGGTGGCTGTTATAATG
Mfd13  (CA)4CGCG(CA)19C                 TTCCCTTTGCTCCCCAAACG: ATTAATCCATCTAAAAGCGAA
Mfd14  (AC)23A                          AAGGATATTGTCCTGAGGA: TTCTGATATCAAAACCTGGC
Mfd15  (AC)25                           GGAAGAATCAAATAGACAAT: GCTGGCCATATATATATTTAAACC
Mfd16  G(CG)4(CA)5TA(CA)3(TA)2(CA)6     AGAGATTAAAGGCTAAATTC: TTCGTAGTTGGTTAAAATTG
       CCAA(CA)21

Mfd17  (AC)23                           TTTCCACTGGGGAACATGGT: ACTCTTTGTTGAATTCCCAT
Mfd18  (AC)18                           AGCTATCATCACCCTATAAAAT: AGTTTAACCATGTCTCTCCCG
Mfd19  (AC)8AG(AC)3AG(AC)24             TCTAACCCTTTGGCCATTTG: GCTTGTTACATTGTTGCTTC
       TCAC(TC)6T
Mfd20  (AC)17                           TTTGAGTAGGTGGCATCTCA: TTAAAATGTTGAAGGCATCTTC
Mfd21  (TA)6TT(TA)2TC(TA)5TT(TA)3       GCTCAGGAGTTCGAGATCA: CACCACACCCGACATTTTA
       CA(TA)7(CA)8TACATG(TA)3
Mfd22  (TA)4(CATA)2(CA)20(GA)2(CAGA)5   TGGGTAAAGAGTGAGGCTG: GGTCCAGTAAGAGGACAGT
Mfd23  (AC)20                           AGTCCTCTGTGCACTTTGT: CCAGACATGGCAGTCTCTA
Mfd24  (AC)7AGAG(AC)14 A                AAGCTTGTATCTTTCTCAGG: ATCTACCTTGGCTGTCATTG
Mfd25  (CA)11                           TTTATGCGAGCGTATGGATA: CACCACCATTGATCTGGAAG
---------------------------------------------------------------------------------------
</TABLE>

Example II

In this example a subset of the sequences isolated and identified as in Example
I were amplified and labeled using the polymerase chain reaction, and were then
resolved on polyacrylamide gels to demonstrate length polymorphisms in these
sequences.
<PAGE>

General Procedures

Oligodeoxynucleotide primers were synthesized on a Cyclone DNA synthesizer
(Biosearch, Inc., San Rafael, Calif. Primers were 19-22 total bases in length,
and contained 7-11 G+C bases. Self-complementary regions in the primers were
avoided.

Genomic DNA was isolated from nucleated blood cells as described (Aldridge et al
(1984), Am. J. Hum. Genet. 36:546-564). Standard polymerase chain reactions
(Saiki et al (1985), Science 230:1350-1354; Mullis and Faloona (1987), Method
Enzymol. 155:335-350; Saiki et al (1988), Science 239:487-491) were carried out
in a 25 (mu)l volume containing 10-20 ng of genomic DNA template, 100 ng each
oligodeoxynucleotide primer, 200 (mu)M each dGTP, dCTP and dTTP, 2.5 (mu)M dATP,
1-2 (mu)Ci of alpha 32 P-dATP at 800 CI/mmole or alpha (35) S-dATP at 500
Ci/mmole, 50 mM KCl, 10 mM Tris, pH 8.3, 1.5 mM MgCl2, 0.01% gelatin and about
0.75 unit of Taq polymerase (Perkin Elmer Cetus, Norwalk, Conn.). Samples were
overlaid with mineral oil and processed through 25 temperature cycles consisting
of 1 min at 94 degree. (denaturation), 2 min at 55degree (annealing), and 2.5
min at 72 degree (elongation). The last elongation step was lengthened to 10
min.

Results shown in FIG. 1 were obtained using conditions slightly different than
the standard conditions. Templates were 100-200 ng of genomic DNA, annealing
steps were 2.5 min at 37degree, elongation steps were 3.5 min at 72 degree, and
alpha (35) S-dATP was added after the 18th cycle rather than at the beginning of
the reactions. The plasmid DNA sample was amplified starting with 50 pg of total
plasmid DNA as template.

Primers for the Mfd15 marker shown in FIG. 2 are listed in Table 1. Primers for
the Mfd26 marker also shown in FIG. 2 are CAGAAAATTCTCTCTGGCTA and
CTCATGTTCCTGGCAAGAAT, and primers for the Mfd31 marker are TAATAAAGGAGCCAGCTATG
and ACATCTGATGTAAATGCAAGT.

Aliquots of the amplified DNA were mixed with two volumes of formamide sample
buffer and electrophoresed on standard denaturing polyacrylamide DNA sequencing
gels. Gels were then fixed, dried and processed for autoradiography. Exposure
times were about 2 days. Gel size standards were dideoxy sequencing ladders
produced using m13, mp10 or mp8 DNA as template.

Results

FIG. 1 shows the amplified DNA fragments for the IGF1 marker in seven unrelated
individuals (1-7). Z represents the most frequent allele; Z-2, the allele that
is two bp larger than the most frequent; Z-2, the allele that is 2 bp smaller,
etc. K indicates Kpn I digestion of amplified samples 1 and 7. Kpn digestions
reduce the number of bands to half the original number because the CA strand,
which normally migrates with an apparent size of about four bases less than the
GT strand, is after Kpn I digestion, four bases longer than the GT strand
resulting in comigration of the two strands. P refers to DNA amplified from a
plasmid DNA sample containing the IGF1 (CA)n block. Sizes of the DNA fragments
in bases are indicated on the left. At the top of the figure are shown the
sequence of the amplified DNA along with the primer sequences and the site of
Kpn I cleavage.

Because the CA and GT strands of the amplified DNA fragments migrate with
different mobilities under the denaturing electrophoresis conditions (see
Example III below), homozygotes yield two bands
<PAGE>

and heterozygotes four bands. The band corresponding to the faster moving CA
strand is more intense on the autoradiographs than the band for the slower GT
strand because the adenine content of the CA strand is higher and labeling is
with alpha (35) S-dATP. Two of the seven individuals shown in FIG. 1 (1 and 3)
were homozygous for the predominant allele (Z) of the IGF1 (CA)n block; the
remainder were heterozygotes of various types.

Proof that the amplified DNA was really from the IGF1 gene and not from some
other portion of the genome includes; that the amplified DNA was of the general
expected size range for the primers used, that the amplified DNA hybridized to
nick-translated poly(dC-dA).poly(dG-dT) (not shown), that this DNA was cleaved
by a restriction enzyme, Kpn I, at the expected position (FIG. 1, lanes 1K and
7K), and that plasmid DNA containing the IGF1 sequence could be used as
polymerase chain reaction template to yield DNA of the same size as was
amplified from the genomic DNA templates (FIG. 1, lane P).

FIG. 2 shows additional examples of polymorphic amplified DNA fragments
containing (dC-dA)n.(dG-dT)n sequences. In this case three different markers
fragments, Mfd15, Mfd26 and Mfd31 were amplified simultaneously from genomic DNA
templates from several different individuals.

Example III

Comparison of different labeling approaches.

General Procedure

The ApoAII (Mfd3) CA or GT strand oligodeoxynucleotide primers were end-labeled
for 1 h at 37.degree. in a 50 .mu.l reaction containing 90 pmoles (600 ng) of
primer, 33 pmoles of gamma (32) P-ATP at 3000 Ci/mmole, 10 mM MgCl2, 5 mM DTT,
50 mM Tris, pH 7.6, and 50 units of T4 polynucleotide kinase. Polymerase chain
reactions were carried out in 25 (mu)l volumes with 50 ng of end-labeled primer
and 86 ng of each unlabeled primer. Interior labeling was performed as in
Example II.

Results

Rather than labeling the amplified DNA throughout the interiors of both strands,
one or both of the polymerase chain reaction primers can be end-labeled using
polynucleotide kinase. FIG. 3 shows the results of such an experiment using as
template, DNA from three different individuals (1-3) and labeling throughout the
interiors of both strands versus labeling of the GT strand primer only versus
labeling of the CA strand primer only. Individual 1 is a homozygote and
individuals 2 and 3 are heterozygotes. Note that because of strand separation
during the denaturing gel electrophoresis labeling of both strands produces two
major bands per allele on the autoradiograph, whereas labeling of the GT strand
primer gives predominantly only the upper band for each allele and labeling the
CA strand primer gives predominantly only the lower band for each allele.
Additional fainter bands on the autoradiograph are artifacts of the polymerase
chain reaction and will be discussed in Example VI.

Example IV

Estimates of informativeness and allele frequencies for the (dC-dA)n.(dG-dT)n
markers.
<PAGE>

General Procedure

Estimates of PIC (polymorphism information content) (Botstein et al (1980),
Amer. J. Hum. Genet. 32:314-331) and heterozygosity were obtained by typing DNA
from 41-45 unrelated Caucasians for markers Mfd1-Mfd4, or by typing DNA From
75-78 parents of the 40 CEPH (Centre d'Etude du Polymorphisme Humain, Paris,
France) reference families for markers Mfd5-Mfd10. The CEPH families are from
the U.S.A., France, and Venezuela. Estimates of allele frequencies were
calculated from the same data.

Results

PIC and heterozygosity values for the first ten (dC-dA)n.(dG-dT)n markers are
shown in Table 2. Values ranged from 0.31 to 0.80 with average PIC of
0.54.+-.0.14 and average heterozygosity of 0.56.+-.0.15. The informativeness of
the (CA)n block markers is generally superior to standard unique sequence probe
polymorphisms (Gilliam et al (1987), Nucleic Acids Res. 15:1445-1458; Overhauser
et al (1987), Nucleic Acids Res. 15:4617-4627; Schumm et al (1988), Am. J. Hum.
Genet. 42:143-159) and as good as many minisatellite polymorphisms (Nakamura et
al (1987), Science 235:1612-1622). Considering the vast number of (CA)n blocks
in the human genome, it is likely that a subset of up to several thousand can be
identified with average heterozygosities of 70% or better.

The number of different alleles detected for the first ten markers (Table 2)
ranged from 4 to 11. Alleles always differed in size by multiples of two bases
(from CA strand to CA strand bands), consistent with the concept that the number
of tandem dinucleotide repeats is the variable factor. Allele frequencies for
the first ten markers are shown in Table 3. For most of the test markers, major
alleles were clustered in size within about 6 bp on either side of the
predominant allele. Amplified fragments must be small enough so that alleles
differing in size by as little as two bases can easily be resolved on the
polyacrylamide gels. The size differences between the largest and smallest
alleles were <20 bp for most of the markers, and therefore several markers can
be analyzed simultaneously on the same gel lane (see FIGS. 2 and 4).

TABLE 2
================================================================================
                  INFORMATIVENESS OF (DC-DA)n.(DG-DT)n MARKERS

                      Length of                      Number
Marker   Chromosome   Amplified        Repeat          of       Hetero-
 (a)     Location       DNA(b)       Sequence(c)     Alleles   zygosity   PIC(d)
--------------------------------------------------------------------------------
Mfd1     12q22-q24.1    192 bp       CATA(CA)19         5         54%      0.53
Mfd2     3q21-qter      120 bp     (CA)13A(CA)17        4         34%      0.31
Mfd3     1q21-q23       137 bp         (AC)16           6         74%      0.65
Mfd4     3q28           169 bp   (AC)12GCACAA(AC)13     6         51%      0.46
Mfd5     19q12-q13.2    151 bp      (CT)7(CA)23        11         80%      0.79
Mfd6          --        192 bp     (CA)5AA(CA)13        7         49%      0.50
Mfd7          --        213 bp     (CA)20TA(CA)2        6         54%      0.51
Mfd8          --        185 bp         (AC)20           8         58%      0.58
Mfd9     19e            100 bp         (CA)17           9         72%      0.69
Mfd10    19             138 bp         (AC)14           6         39%      0.42

--------------------------------------------------------------------------------

(a) Mfd stands for Marshfield. The marker name substitutes for the probe name in
    RFLP markers and denotes a specific pair of amplification primers for each
    locus.
<PAGE>

(b) Sizes of amplified DNA fragments corresponded to the predominant allele for
    each marker. Estimated error is 2 bp.

(c) Sequences for Mfd1-Mfd5 were taken from GenBank. Sequences for Mfd6-Mfd10
    were determined in the laboratory.

(d) PIC is Polymorphism Information Content (Botstein et al (1980), Am J. Hum.
    Genet. 32:314-331).

(e) Markers 9 and 10 were developed using clones selected from a chromosome
    19specific phage library.

                                     TABLE 3

--------------------------------------------------------------------------------
                Allele Frequencies for (dC-dA)n.(dG-dT)n Markers

                                              Marker
                            ------------------------------------------
ALLELEA         1      2     3      4      5      6      7      8      9     10
               ---    ---   ---    ---    ---    ---    ---    ---    ---    ---
Z + 14         --(b)  --    --     --      1%    --     --     --     --     --
Z + 10         --     --    --     --     --      3%    --      1%    --     --
Z + 8          --     --     3%    --      3%    11%    --      3%    --     --
Z + 6          --     --    35%     2%     4%     1%    --      7%     3%     1%
Z + 4          18%     6%   --      6%    15%     1%     6%     2%     1%    --
Z + 2          16%    11%    1%    13%    15%     3%     4%    27%    12%     5%
Z              60%    81%   39%    70%    34%    64%    66%    54%    38%    73%
Z - 2           5%     2%   --      1%     3%    18%    15%     4%     6%    --
Z - 4          --     --     8%    --      2%    --      7%     1%     4%    10%
Z - 6          --     --    14%     8%     1%    --     --     --      4%     9%
Z - 8          --     --    --     --     --     --      2%    --     31%     1%
Z - 12         --     --    --     --     --     --     --     --      1%    --
Z - 14         --     --    --     --     11%    --     --     --     --     --
Z - 16          1%    --    --     --     --     --     --     --     --     --
Z - 22         --     --    --     --     12%    --     --     --     --     --
SPREAD(c)(bp)  20      6    14     12     36     12     12     14     18     14
--------------------------------------------------------------------------------

(a) Z indicates the predominant allele for each marker.

(b) A dash means this allele was not found.

(c) Spread is the size difference between amplified DNA fragments corresponding
    to largest and smallest alleles.

Example V

Demonstration of Mendalian codominant inheritance of (dC-dA)n.(dG-dT)n markers.

General Procedure

DNA from individuals of the CEPH families and from other three generation
families was used as template for the amplification of various (dC-dA)n.(dG-dT)n
markers using the procedure described in Example II.

Results

FIG. 4 shows the amplified DNA from markers Mfd1, Mfd3 and Mfd4 from CEPH family
1423. DNA fragment sizes in bases are marked to the left of the gel. Individual
genotypes are listed below the gel. All three markers showed Mendelian behavior
for this family.

A total of approximately 500 family/marker combinations have been tested to
date. Mendelian codominant inheritance has been observed in all cases; no new
mutations have been found. Therefore new mutations are unlikely to be a general
problem with the (dC-dA)n.(dG-dT)n markers.
<PAGE>

Example VI

Artifacts of the amplification reactions.

General Procedure

Aliquots of two different (dC-dA)n.(dG-dT)n amplified fragments (two different
markers from two individuals), untreated from the polymerase chain reaction (C),
were brought up to 200 (mu)M dATP and then incubated at 37.degree. for 30 min
with 6 units of Klenow enzyme (K), 1 unit of T4 DNA polymerase (P), or with no
additional enzyme (T). Samples were then mixed with formamide sample buffer and
loaded on polyacrylamide gels.

For the results shown in FIG. 6, DNA from an individual with the Mfd3 genotype
Z+6, Z-6 was amplified with modified Mfd3 primers (AGGCTGCAGGATTCACTGCTGTGGACCCA
and GTCGGTACCGGTCTGGAAGTACTGAGAAA) so that sites for the restriction enzymes Pst
I and Kpn I were located at opposite ends of the amplified DNA. An aliquot of
the amplified DNA from a 27 cycle reaction was diluted 60,000 fold with 0.2X TE,
and 10 (mu)l of the dilution (approximately 10.sup.5 molecules) were amplified
with the same primers for another 27 cycles. An aliquot of the second
amplification reaction was diluted as above and subjected to a third 27 cycle
reaction. Amplified DNA samples were treated with T4 DNA polymerase prior to
electrophoresis.

Amplified DNA from the first reaction described in the above paragraph was
digested with Kpn I and Pst I, extracted with phenol, and simultaneously
concentrated and dialyzed into 0.2X TE using a Centricon 30 cartridge. This DNA
was then ligated into mp19 and transformed into E. coli. Clear plaques on
X-gal/IPTG plates from the transformed cells were picked, amplified and used to
isolate single-stranded DNA. DNA from 102 such clones was sequenced. The
distribution of the numbers of dinucleotide repeats in these clones is shown in
Table 4.

Results

Additional bands, less intense than the major pair of bands for each allele and
smaller in size than the major bands, were usually seen for the amplified DNA
fragments. These bands are particularly apparent in FIGS. 2-4. The additional
bands were present when cloned DNA versus genomic DNA was used as template (FIG.
1, lane P), and even when such small amounts of heterozygote genomic DNA were
used as template that only one of the two alleles was amplified. Also, DNA
amplified from 63 lymphocyte clones (gift of J. Nicklas) (Nicklas et al (1987),
Mutagenesis 2:341-347) produced from two individuals showed no variation in
genotype for all clones from a single donor. These results indicate that the
additional bands are generated as artifacts during the amplification reactions,
and are not reflections of somatic mosaicism.

Griffin et al. (1988), (Am. J. Hum. Genet. 43(Suppl.):A185) recently
demonstrated that DNA fragments amplified by the PCR could not be efficiently
ligated to blunt ended vectors without first repairing the ends of the fragments
with T4 DNA polymerase. To test whether "ragged" ends were responsible for the
extra bands associated with (dC-dA)n.(dG-dT)n amplified fragments, amplified DNA
(C) was treated with the Klenow fragment of DNA polymerase I (K), with T4 DNA
polymerase (P) or with no enzyme (T) as shown in FIG. 5. Both Klenow enzyme and
T4 DNA polymerase simplified the banding
<PAGE>

pattern somewhat by eliminating extra bands which differed in size from the most
intense bands by 1 base. These enzymes also reduced the size of the most intense
bands by 1 base. The most likely explanation for these results is the Taq
polymerase produces a mixture of fragments during the PCR with different types
of ends. The most intense bands in the untreated samples are likely derived from
double stranded molecules with single base noncomplementary 3' overhangs. The
fainter bands which are 1 base smaller than the major bands are likely to be
blunt ended. The 3'-5' exonuclease activity of Klenow or T4 DNA polymerase
converts the molecules with overhangs into blunt ended molecules. Clark (1988),
(Nucleic Acids Res. 16:9677) recently showed that a variety of DNBA polymerases
including Taq polymerase can add a noncomplementary extra base to the 3' ends of
blunt ended molecules.

Remaining after Klenow or T4 DNA polymerase treatment are extra bands which
differ in size from the major bands by multiples of two bases. The data in Table
4 and FIG. 6 strongly indicate that these particular extra bands are the result
of the skipping of repeats by the Taq polymerase during the amplification
cycles. Sequencing of individual clones of DNA amplified for 27 cycles (first
lane, FIG. 6) verifies that repeats have been deleted in the amplified DNA
(Table 4). The largest of the predominant-sized fragments in the amplified DNA
(with the exception of the one 20-mer) are 19 and 13 repeats in length,
consistent with the Z+6, Z-6 genotype of the donor of the template DNA.
Substantial numbers of clones containing fewer repeats are also seen, and this
distribution matches the pattern of fragments shown in the first lane of FIG. 6.
If the Taq polymerase is skipping repeats during the amplification cycles, as
seems likely from the sequencing data, then further cycles of amplification in
addition to the first 27 should reduce the intensities of the bands
corresponding to the original DNA in the template and increase the intensities
of the bands corresponding to fragments with skipped repeats. This is exactly
what is observed in FIG. 6 for the amplified DNA from the reactions with 54 and
81 total amplification cycles.

                                     TABLE 4

--------------------------------------------------------------------------------
          Distribution of numbers of repeats in clones of amplified DNA

           NUMBER OF REPEATS                      NUMBER OF CLONES
--------------------------------------------------------------------------------
                  20                                      1
                  19                                     20
                  18                                     22
                  17                                      4
                  16                                      5
                  15                                      1
                  14                                      0
                  13                                     22
                  12                                     18
                  11                                      3
                  10                                      3
                   9                                      3
--------------------------------------------------------------------------------

Example VII

Use of (dC-dA)n.(dG-dT)n polymorphisms to identify individuals.
<PAGE>

General Procedure

Genomic DNA from a collection of 18 unrelated individuals and from an unknown
individual was isolated from blood and amplified with various (dC-dA)n.(dG-dT)n
markers as described under Example II. Genotype frequencies were calculated from
the allele frequencies shown in Table 3 assuming Hardy-Weinberg equilibria.

Results

One individual out of a group of 18 unrelated volunteers was selected so that
the identity of this individual was unknown. The unknown DNA sample and the
control samples were then typed for the Mfd3 and Mfd4 markers. As shown in Table
5, only three of the 18 controls had Mfd3 and Mfd4 genotypes consistent with the
unknown sample, namely, individuals 22, 35 and 42. Further typing of these three
samples and the unknown with four additional markers as shown in Table 5,
conclusively demonstrated that the unknown DNA sample came from individual 22.

Table 6 shows the expected genotype frequencies for the six typed markers for
individual 22 using the allele frequency data from Table 3. Single genotypes
range in frequency from 0.04 to 0.49. The frequency for the entire collection of
six markers is the product of the individual probabilities or
1.5.times.10.sup.-5 or about 1 in 65,000 people. Choosing a better, more
informative collection of (dC-dA)n.(dG-dT)n markers would result in considerably
greater discrimination.

                                     TABLE 5

--------------------------------------------------------------------------------
                        Genotypes for identification test
                        ---------------------------------
                                     Markers
                  ---------------------------------------------

INDIV.    MFD3         MFD4        MFD9          MFD1         MFD8        MFD11
--------------------------------------------------------------------------------
  7      137,137     169,171
  8      131,137     169,169
 10      131,143     169,169
 11      131,143     169,163
 12      143,133     169,173
 14      137,143     169,169
 22      137,137     169,169      98,92        194,192      187,185      108,116
 ukn     137,137     169,169      98,92        194,192      187,185      108,116
 23      139,143     169,167
 25      137,143     169,163
 26      133,143     169,171
 27      143,143     169,169
 30      137,143     169,171
 31      137,143     169,169
 33      137,143     169,171
 34      143,143     169,169
 35      137,137     169,169      100,92       192,192      185,183      108,108
 41      137,131     169,171
 42      137,137     169,169      104,100      192,192      185,185      120,108
--------------------------------------------------------------------------------
<PAGE>

                                     TABLE 6

--------------------------------------------------------------------------------
                              Genotype frequencies
                              --------------------
                      Genotype              Allele                 Genotype*
Marker                for #22          Frequency for #22       Frequency for #22
--------------------------------------------------------------------------------
  Mfd3                137,137          39% 137                        15%
  Mfd4                169,169          70% 169                        49%
  Mfd9                  98,92          6% 98, 31% 92                   4%
  Mfd1                192,194          60% 192, 16% 194               20%
  Mfd8                185,187          54% 185, 27% 187               29%
  Mfd11               108,116          43% 108, 10% 116                9%
                                                                      --
                                            Total =               0.0015%

--------------------------------------------------------------------------------

*Homozygote genotype frequency = (allele frequency)(2)
Heterozygote genotype frequency = 2(allele1)(allele(2))

Various modes of carrying out the invention are contemplated as being within the
scope of the following claims particularly pointing out and distinctly claiming
the subject matter which is regarded as the invention.

  I claim:

1. A method of analyzing or typing polymorphic DNA fragments which contain
repeats which are specific to particular loci in the genome comprising:
amplifying at least one polymorphic DNA fragment containing at least one
(dC-dA)n.(dG-dT)n segment using the polymerase chain reaction with a DNA sample
which includes the said DNA fragment as a template, and with two oligonucleotide
primers sufficiently complementary to non-repeated sequences at the ends of said
fragment within said sample to hybridize therewith, and separating and
characterizing amplified fragments on the basis of size.

2. The method of claim 1 further comprising labeling or marking said polymorphic
DNA fragments.

3. The method of claim 1 wherein said primers are obtained by direct synthesis.

4. The method of claim 1 wherein said primers are end-labeled.

5. The method of claim 1 wherein more than one DNA fragment containing at least
one segment of (dC-dA)n.(dG-dT)n is amplified and characterized simultaneously.

6. The method of claim 1 used to establish the identity, pedigree, or
relationship of an individual comprising comparison of said amplified fragments
with amplified fragments from the DNA of one or more control individuals wherein
the control DNA samples have 25 been similarly amplified and characterized.

7. The method of claim 1 wherein the amplified fragments are separated and
characterized by gel electrophoresis.

8. The method of claim 1 wherein the DNA sample is 30 genomic DNA.

9. The method of claim 1 wherein the DNA sample is cloned DNA.

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