Biotechnology �By�Ms. Rabbiah Manzoor

Biotechnology and Human Disease

• Restriction endonucleases
• DNA cloning
• Probes
• Southern blotting
• Restriction fragment length polymorphism
• Polymerase chain reaction
• Analysis of gene expression

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Learning Outcomes

• Write a note on PCR& Southern blotting techniques
• Explain Probes
• Explain Prenatal Diagnosis
• Discuss Gene therapy & gene expression
• Summarize DNA Cloning
• Explain Restriction fragment length polymorphism

Restriction endonucleases

• These are the special group of enzymes which cleave double stranded DNA into smaller fragments
• Each enzymes cleaves DNA at specific nucleotide sequence
• These enzymes are used experimentally to obtain precisely defined DNA segments called restriction fragments

Specificity of�Restriction Endonucleases

• These enzyme recognize short stretches of DNA (4-8 bp) that contain specific nucleotide sequences.
• These sequences are palindromes

In bacteria restriction endocucleases restrict the expression of non bacterial DNA through cleavage. Bacterial DNA is protected by methylation of bases at the restriction sites

Nomenclature of�Restriction Endonucleases

• A restriction enzyme is named according to the organism from which it was isolated.
• The first letter of the name is from the genus of the bacterium.
• The next two letters are from the name of the species
• An additional letter indicates the type of strain and a final number to indicate the order in which the enzyme was discovered in that particular organism
• HaeIII- third enzymes from Haemophilus aegyptius

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Restriction sites

• A DNA sequence that is recognized and cut by a restriction enzyme is called restriction site
• An enzyme that recognizes four base pair sequence produces many cuts in DNA
• And the enzyme detecting six bp long sequence produces fewer fragments

DNA CLONING

• Defined as production of many identical copies of DNA
• Can be done by introducing a foreign DNA molecule into a replicating cell
• Human DNA can be obtained from blood, saliva and solid tissues for cloning
• Important terms for cloning
• Vectors
• DNA libraries
• Clonned DNA fragments

PROBES

• A short piece of ssDNA labeled with a radioisotope such as P³² or with a non radioactive molecule such as biotin
• Sequence of probe is complementary to the sequence in the DNA of interest called the target DNA
• Probes are used to identify which band on gel or which clone contains the target DNA, this process is called screening

Southern blotting

• Technique forfor detecting mutations in DNA
• Combination of
• Restriction enzymes
• Electrophoresis
• DNA Propbes

Procedure

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1) DNA (genomic or other source) is digested with a restriction enzyme and separated by gel electrophoresis, usually an agarose gel. Because there are so many different restriction fragments on the gel, it usually appears as a smear rather than discrete bands. The DNA is denature into single strands by incubation with NaOH.

2) The DNA is transfered to a membrane which is a sheet of special blotting paper. The DNA fragments retain the same pattern of separation they had on the gel.

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Procedure

3) The blot is incubated with many copies of a probe which is single-stranded DNA. This probe will form base pairs with its complementary DNA sequence and bind to form a double-stranded DNA molecule. The probe cannot be seen but it is either radioactive or has an enzyme bound to it (e.g. alkaline phosphatase or horseradish peroxidase).

4) The location of the probe is revealed by incubating it with a colorless substrate that the attached enzyme converts to a colored product that can be seen or gives off light which will expose X-ray film. If the probe was labeled with radioactivity, it can expose X-ray film directly.

Mutations and Polymorphism

• The genomes of nonrelated people differ at about one in 1200 DNA bases, or about 1% of genome
• These genome variations could be mutation or polymorphism
• Mutation refers to an infrequent but potentially harmful genome variation
• Polymorphism is a clinically harmless DNA variation that does not affect the phenotype
• It primarily occurs in those regions of the DNA which do not code for a protein

Restriction Fragment Length Polymorphism

• Prenatal diagnosis of sickle cell anemia
• Phenylketoniuria

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Gene Expression analysis�Gene Therapy

• Study the content given in Lipincott’s Biochemistry