DNA Cloning: The Basics:- Alterations in Cloned Genes Produce Modified Proteins

Cloning techniques can be used not only to overproduce proteins but to produce protein products subtly altered from their native forms. Specific amino acids may be re placed individually by site-directed mutagenesis. This powerful approach to studying protein structure and function changes the amino acid sequence of a protein by altering the DNA sequence of the cloned gene. If appropriate restriction sites flank the sequence to be altered, researchers can simply remove a DNA segment and replace it with a synthetic one that is identical to the original except for the desired change (Fig. 9–12a). When suitably located restriction sites are not present, an approach called oligonucleotide-directed muta genesis (Fig. 9–12b) can create a specific DNA sequence change. A short synthetic DNA strand with a specific base change is annealed to a single-stranded copy of the cloned gene within a suitable vector. The mismatch of a single base pair in 15 to 20 bp does not prevent annealing if it is done at an appropriate temperature. The annealed strand serves as a primer for the synthesis of a strand complementary to the plasmid vector. This slightly mismatched duplex recombinant plasmid is then used to transform bacteria, where the mismatch is repaired by cellular DNA repair enzymes. About half of the repair events will re move and replace the altered base and restore the gene to its original sequence; the other half will remove and replace the normal base, retaining the desired mutation. Transformants are screened (often by sequencing their plasmid DNA) until a bacterial colony containing a plasmid with the altered sequence is found.

Changes can also be introduced that involve more than one base pair. Large parts of a gene can be deleted by cutting out a segment with restriction endonucleases and ligating the remaining portions to form a smaller gene. Parts of two different genes can be ligated to create new combinations. The product of such a fused gene is called a fusion protein. Researchers now have ingenious methods to bring about virtually any genetic alteration in vitro. Reintroduction of the altered DNA into the cell permits investigation of the consequences of the alteration. Site directed mutagenesis has greatly facilitated research on proteins by allowing investigators to make specific changes in the primary structure of a protein and to ex amine the effects of these changes on the folding, three dimensional structure, and activity of the protein.

اخر الاخبار

اخبار العتبة العباسية المقدسة

وفد العتبة العباسية المقدسة يزور شعبة الإعداد البدني للاطّلاع على برنامج تأهيل المنتسبين الجدد

قسم بين الحرمين يقدم جهودًا مضاعفة لإعادة المفقودات إلى الزائرين

بعد برامج تدريبية متخصصة.. متحف الكفيل يبيّن منجزات ملاكاته في فن الخاتم (الأرابيسك)

شعبة مدارس الكفيل الدينية تعقد الاجتماع التحضيري الأول لمهرجان روح النبوّة الثقافي

المزيد

الآخبار الصحية

ماذا يحدث إذا تناولنا الطعام مرة واحدة فقط في اليوم

اكتشاف عوامل خطر غير متوقعة لمرض الكبد الدهني

فيروس هانتا والسفينة الموبوءة.. تحديث من الصحة العالمية

طبيب يحسم الجدل السجائر الإلكترونية وعلاقتها بالسرطان

المزيد

الآخبار التكنلوجية

لأول مرة في الولايات المتحدة.. تشغيل مفاعل مصغر نووي يغذي الذكاء الاصطناعي بالطاقة

الكشف عن تفاصيل أول عملية ناجحة لاستعادة انتاج الحيوانات المنوية

أطلس يكشف خفايا الجسم البشري بدقة أرفع بـ50 مرة من شعر الإنسان

المتحف المصري الكبير يتحول إلى "أيقونة خضراء" بدعم ياباني

المزيد

مواضيع ذات صلة

Storage Lipids: - Triacylglycerols Are Fatty Acid Esters of Glycerol

Storage Lipids:- Fatty Acids Are Hydrocarbon Derivatives

Genome Alterations and New Products of Biotechnology: -Recombinant DNA Technology Yields New Products and Challenges

Genome Alterations and New Products of Biotechnology:- New Technologies Promise to Expedite the Discovery of New Pharmaceuticals

Genome Alterations and New Products of Biotechnology: -Manipulation of Animal Cell Genomes Provides Information on Chromosome Structure and Gene Expression

Genome Alterations and New Products of Biotechnology:- A Bacterial Plant Parasite Aids Cloning in Plants

From Genomes to Proteomes:- Detection of Protein-Protein Interactions Helps to Define Cellular and Molecular Function

From Genomes to Proteomes:- Cellular Expression Patterns Can Reveal the Cellular Function of a Gene

From Genomes to Proteomes:- Sequence or Structural Relationships Provide Information on Protein Function

From Genes to Genomes:- Genome Sequences Provide the Ultimate Genetic Libraries

From Genes to Genomes:- The Polymerase Chain Reaction Amplifies Specific DNA Sequences

From Genes to Genomes:- DNA Libraries Provide Specialized Catalogs of Genetic Information