النبات
مواضيع عامة في علم النبات
الجذور - السيقان - الأوراق
النباتات الوعائية واللاوعائية
البذور (مغطاة البذور - عاريات البذور)
الطحالب
النباتات الطبية
الحيوان
مواضيع عامة في علم الحيوان
علم التشريح
التنوع الإحيائي
البايلوجيا الخلوية
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البكتيريا
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علم الأمراض
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اضطرابات الدورة الدموية
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التقانة الإحيائية
مواضيع عامة في التقانة الإحيائية
التقنية الحيوية المكروبية
التقنية الحيوية والميكروبات
الفعاليات الحيوية
وراثة الاحياء المجهرية
تصنيف الاحياء المجهرية
الاحياء المجهرية في الطبيعة
أيض الاجهاد
التقنية الحيوية والبيئة
التقنية الحيوية والطب
التقنية الحيوية والزراعة
التقنية الحيوية والصناعة
التقنية الحيوية والطاقة
البحار والطحالب الصغيرة
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مفاهيم التقنية الحيوية النانوية
التراكيب النانوية والمجاهر المستخدمة في رؤيتها
تصنيع وتخليق المواد النانوية
تطبيقات التقنية النانوية والحيوية النانوية
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المصفوفات المجهرية وحاسوب الدنا
اللقاحات
البيئة والتلوث
علم الأجنة
اعضاء التكاثر وتشكل الاعراس
الاخصاب
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العصيبة وتشكل الجسيدات
تشكل اللواحق الجنينية
تكون المعيدة وظهور الطبقات الجنينية
مقدمة لعلم الاجنة
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مواضيع عامة في الاحياء الجزيئي
علم وظائف الأعضاء
الغدد
مواضيع عامة في الغدد
الغدد الصم و هرموناتها
الجسم تحت السريري
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الغدة الصنوبرية
مواضيع عامة في علم وظائف الاعضاء
الخلية الحيوانية
الجهاز العصبي
أعضاء الحس
الجهاز العضلي
السوائل الجسمية
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مضادات الفايروسات
علم الخلية
الوراثة
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المناعة
التحليلات المرضية
الكيمياء الحيوية
مواضيع متنوعة أخرى
الانزيمات
Chromosome Mapping
المؤلف:
Robert Schleif
المصدر:
Genetics and Molecular Biology
الجزء والصفحة:
2nd Edition , p307-310
2025-06-11
59
It is simplest first to determine a large-scale and coarse map of a chromosome and then to build upon the initial map to increase resolution. Construction of a low-resolution physical map can begin with a library of overlapping clones. Mapping would then be picking and ordering a subset of these which spans an entire chromosome. The larger the clones, the fewer which need to be ordered, and the easier it is to construct of the initial map. The yeast artificial chromosome vectors, YAC vectors, are useful for chromosome mapping projects because they can accommodate very large fragments of foreign DNA, up to 106 base pairs. These contain autonomous replicating sequences, ARS, as origins and telomeres for the ends.
Let us first consider the physical markers which are necessary for the construction of a physical map. Unique sequences of DNA one or two hundred base pairs long can provide a good starting point for construction of a physical map. Such sequences can be obtained by sequencing random clones made from the chromosome to be mapped. The unique ness of a sequence can be checked by hybridization. From such a unique sequence, two 20 to 30 base portions can be chosen for oligonucleotide primers that will permit PCR amplification of the sequence lying between. This amplification will occur only if the template DNA present in the PCR reaction contains the unique sequence. Such a unique
sequence is called a sequence-tagged site, or STS. A sample of DNA is tested for the presence of an STS by PCR amplification using the two oligonucleotides followed by gel electrophoresis to assay for the amplification of the proper-sized DNA fragment.
When the number of sequence-tagged sites exceeds the number of clones necessary to span the chromosome by a factor of four or five or more, the sites can be used just like restriction enzyme cleavage sites are used in a chromosomal walk. That is, each YAC vector is assayed for the presence of each STS. If an STS site is present in two vectors, then the chromosome fragment carried by each contains this region in common (Fig. 1). Merely from this data, the STS sites can be ordered and an overlapping physical map of the chromosome can be constructed. Of course, for even the smallest human chromosome, tens of thousands of YAC vectors must be present in the library, and hundreds of STS sites are needed. Therefore, very many PCR amplification reactions will be required for the ordering.
Fig1. A set of DNA fragments ordered by the STS sites they share in common.
Mutations, insertions, and deletions have also been used in chromo some mapping. Originally, restriction fragment length polymorphisms, RFLPs were used as genetic and physical markers for chromosome mapping. Over the majority of the genome, sequence differences between individuals exist at a frequency of about 10-4. If one of these differences occurs in the cleavage site for a restriction enzyme an RFLP will exist there. Another source of an RFLP is an insertion or deletion or a difference in the number of repeats of a short sequence contained between the two restriction enzyme cleavage sites.
Restriction fragment length polymorphisms can be detected using Southern transfers as described in an earlier section. If individuals differ in one out of 104 base pairs, then the chances that a particular restriction enzyme cleavage site will be present in one chromosome but be missing in the homologous chromosome would be about 0.001. There is a reasonably high probability that either an insertion exists between two restriction enzyme cleavage sites or that two individuals differ at a particular point in the number of times that a short sequence is repeated there. One out of a hundred to one out of a thousand restriction fragments it is likely to be polymorphic between some individuals in the population. Therefore, considerable work could be required to find DNA sequences useful for RFLP mapping. One method for RFLP detection is to clone random segments of human DNA and use them to probe Southern transfers of various enzyme digests from a moderate number of people. The occasional clone which reveals a polymorphism is then mapped to a specific chromosome and finally mapped at higher resolution by comparing its segregation pattern to the pattern of other markers known to lie on the same chromosome.
A more efficient method of finding and mapping genetic markers utilizes the polymerase chain reaction. Instead of using Southern transfers to detect an RFLP, PCR is used to amplify a segment of DNA that contains variable numbers of inserts. Different individuals in the population often possess different numbers of inserts at the same point (Fig. 2). The size of a fragment synthesized by PCR identifies the size of the insert. It is much easier to perform a PCR amplification and determine the size of the product than it is to perform a Southern transfer. Therefore, most chromosome mapping will shift to the use of PCR. By the approaches described above, hundreds of RFLP or PCR based markers have already been mapped to the human genome and DNA defects of a significant number of genetic diseases have been mapped with respect to the markers.
Fig2. A set of DNA fragments ordered by the STS sites they share in common.