Enzim Restriksi dan Fungsinya

Cloning and Recombinant DNA

• Cloning and recombinant DNA are techniques used to transfer DNA fragments between organisms.
• A plasmid, a circular double-stranded DNA molecule, is a vehicle for cloning. Plasmids are often used to transfer antibiotic resistance genes between bacteria.
• Restriction enzymes cut DNA at specific recognition nucleotide sequences, called restriction sites. They're often found in bacteria, acting as a defense mechanism against foreign DNA.
• Restriction enzymes can create blunt ends or sticky ends when cutting DNA. Sticky ends have complementary overhangs, facilitating easier ligation.
• DNA ligase seals the sugar-phosphate backbone of DNA fragments. It's crucial for joining DNA fragments together during recombinant DNA technology.
• Cloning vectors are plasmids or bacteriophages modified to carry foreign DNA. They need an origin of replication, a selectable marker (e.g., antibiotic resistance gene), and multiple cloning sites.
• Types of restriction enzymes are categorized by their composition, enzyme co-factor requirements. and the nature and position of their DNA cleavage sites.
• Isoschizomers have similar recognition sites but different restriction sites.
• Isocaudomers have different recognition sites but similar restriction sites.

Basic Steps in Gene Cloning

• Isolate DNA and fragment it into desired sizes.
• Insert the DNA fragments into a carrier molecule (vector).
• Introduce the recombinant DNA into a host cell (e.g., bacteria).
• Select host cells containing the desired DNA fragment.

Contents

• Cloning principles
• Restriction enzymes
• Host cells
• Vectors
• Promoters
• Transformant selection
• Mobile DNA
• Vector criteria and types

Restriction Enzymes

• Restriction enzymes are molecular scissors that cut DNA at specific sites.
• They act as a protection mechanism against foreign DNA.
• Bacterial endogenous DNA is protected by methylation.
• Recognition sites are short sequences of nucleotides (4-8 base pairs long).
• Many recognition sequences are palindromic.

How Bacteria Prevent Restriction Enzymes

• Bacteria methylate their own DNA to prevent cleavage by restriction enzymes.

Types of Restriction Enzymes

• There are four general groups of restriction enzymes.
  • Type I, II, III, and IV.

Characteristics of Restriction Enzymes

• Type I enzymes cleave DNA at sites far from the recognition sequence. They require ATP and S-Adenosyl-L-methionine.
• Type II enzymes cleave DNA within or close to the recognition sequence. They often require magnesium.
• Type III enzymes cleave DNA at specific distances from the recognition sequence. They need ATP.
• Type IV enzymes cleave modified DNA such as methylated, hydroxymethylated, and glucosyl-hydroxymethylated DNA.

Nomenclature of Restriction Enzymes

• Restriction enzymes are named after the bacteria from which they are purified.
• The name includes the genus, species, and strain.

Restriction Site

• A restriction site is the specific nucleotide sequence recognized by a restriction enzyme.
• Recognition sequences vary in length and many are palindromic (read the same forward and backward on complementary strands).

Palindromic Sequence

• A palindromic sequence reads the same forward and backward on complementary DNA strands.
• The size of the recognition sequence determines how often a restriction enzyme will cut.

Function and Mechanism of Restriction Enzymes

• Restriction enzymes protect cells by restricting the function of infecting DNA.
• They recognize specific sequences and cut DNA, creating blunt ends or sticky ends.

Sticky Ends and Blunt Ends

• Sticky ends are DNA fragments with complementary overhangs.
• Blunt ends are DNA fragments with no overhangs.

Restriction mapping

• A technique to determine the relative locations of different recognition sites of different enzymes in a DNA molecule—cutting with various combinations of restriction enzymes and determining the sizes of fragments.

DNA Ligase

• A molecular glue that joins DNA fragments together.
• Seals the sugar-phosphate backbone of DNA.
• Efficiently seals sticky ends but can also seal blunt ends, though with lower efficiency.
• T4 DNA ligase is commonly used.

Plasmids

• Naturally occurring, extrachromosomal, circular, double-stranded DNA molecules.
• Important for transferring genetic material (e.g., antibiotic resistance) between bacteria.
• Can be modified to carry new genes, which makes them useful cloning vectors.

Cloning Vectors

• Plasmids are commonly used cloning vectors.
• Cloning vectors require an origin of replication.
• A selectable marker.
• Multiple cloning sites.

Chimeric Plasmids

• Combinations of different segments.

Host Cells

• Ideal host cells are easy to handle, propagate, have diverse strains, and accept a variety of vectors.
• Prokaryotes (bacteria) are commonly used in many cloning protocols.
• Eukaryotes are used when the goal is gene expression.
• Bacteria (e.g., E. coli)
• Fungi (e.g., Yeast)
• Plants
• Animals

Prokaryotic Hosts

• E. coli is commonly used for cloning and is well-characterized. But it has limitations on fragment size for effective cloning.
• other bacteria can also be used as hosts.

Eukaryotic Hosts

• Microbes (e.g., yeasts) and more complex organisms (e.g., plants, animals) with specific advantages for particular genetic engineering purposes.

Artificial Chromosomes

• Artificial chromosomes (e.g., YACs and BACs) are used to clone large DNA fragments.
• BACs are more stable than YACs, which helps in cloning large segments of DNA.

Promoter

• Promoter is a specific DNA sequence that dictates where, when, and how much transcription occurs.
• Promoters may be constituitive—always active—or inducible—activated by specific factors.
• Promoters may be tissue-specific, acting only in certain cells or during specific development stages.

Types of Promoters

• Constitutive promoters
• Inducible promoters
• Tissue-specific/developmental-stage specific promoters
• Synthetic promoters

Common Reporter Genes

• Antibiotic-resistance genes
• LacZ gene of E. coli
• GUS reporter gene
• GFP (Green Fluorescent Protein) gene (often isolated from jellyfish)

Bacterial Transformation

• Uptake of foreign DNA by bacteria.
• Selection process often uses antibiotic resistance, since transformed cells are more likely to thrive in an antibiotic-containing medium.

Reporter Gene

• Genes that encode easily detectable proteins.
• Allows scientists to follow gene expression in a specific pathway of cells.

LacZ Gene

• Used as a reporter gene in molecular biology.
• Its product, beta-galactosidase, catalyzes the breakdown of certain substrates, which can be used to identify bacterial cells with the appropriate DNA.

GUS Reporter Gene

• Encodes β-glucuronidase (GUS).
• Compatible with Agrobacterium-mediated transformation.
• Commonly used to analyze promoter activity in plants, because the presence of GUS enzyme is easily tested by visualization.

Fluorescent Protein

• Proteins that fluoresce.
• GFP is a common fluorescent protein used in research.
• Used to analyze promoter activity.

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