BUA Biochemistry II Lab 9 Molecular Biology Techniques PDF

Biochemistry II lab 9 Molecular Biology Techniques cont.  We’ll learn about… 1 2 3 4 5 Restriction Gene Enzymes Southern Western Flow Cloning Cloning Blotting Blotting...

Biochemistry II lab 9 Molecular Biology Techniques cont.  We’ll learn about… 1 2 3 4 5 Restriction Gene Enzymes Southern Western Flow Cloning Cloning Blotting Blotting Cytometry Technology Vectors  Gene Cloning Technology In vivo molecular biology technique that uses a recombinant DNA molecule to create many identical copies of DNA fragment (so called Recombinant DNA technology). Recombinant DNA molecule is a DNA molecule that is generated by combining genetic materials from different sources, using two types of enzymes: - Restriction enzyme (which cut DNA) - DNA ligase (which joins DNA)  Restriction Enzymes ▪ Also called Restriction endonucleases are enzymes made naturally by bacteria as a defense mechanism against bacteriophages (viral DNA). ▪ They recognize specific short DNA sequences (Restriction sequences), and cut the dsDNA at or near these sequences. Recognition Sequence: ▪ Each restriction enzyme has a unique and specific recognition sequence, typically 4-8 base pairs long, that is usually palindromic (read the same forward and backward).  Restriction Enzymes Cutting Action: ▪ The enzyme cuts the DNA molecule by cleaving the phosphodiester bonds between nucleotides in the DNA backbone. ▪ Some enzymes create sticky ends, while others generate blunt ends.  Cloning Vectors ✓ It is a DNA molecule that carries foreign DNA into a host cell where it replicates and produces many copies the foreign DNA. Types of vectors for cloning are : 1. Plasmids: Extra-chromosomal circular double stranded DNA that autonomously replicates inside the bacterial cell. 2. Bacteriophages: Viruses that infect bacteria and multiply inside bacterial cell. 3. Bacterial Artificial Chromosomes (BACS) 4. Yeast Artificial Chromosomes (YACS) Gene Cloning Technology  Southern Blotting ✓ It is a technique for detection of specific DNA fragment using the following steps: Techniques that deal with proteins  1) Western blotting ❖ Also called Immunoblotting, is a technique for the detection and analysis of proteins.  Southern Blotting Northern Blotting Western blotting Immunoblotting Molecule For detection of For detection of For the detection and detected specific DNA specific RNA analysis of proteins Gel SDS-PAGE (Sodium Formaldehyde electrophoresis Agarose gel Dodecyl Sulfate agarose gel matrix Polyacrylamide Gel) Radioactive or non- Radioactive or non- Probe used radioactive Antibody radioactive DNA cDNA or cRNA Restriction Required Not required Not required digestion  2) Flow cytometry A technology that measures and analyzes multiple physical characteristics of single cells, as they flow in a fluid stream through a beam of light. The properties measured include a particle’s relative size, relative granularity, and relative fluorescence intensity. Relative fluorescence intensity can measure cell surface proteins and intracellular protein using specific antibodies against the target protein. The antibody is labeled with a fluorescent dye that can give fluorescence once it stimulated with a beam of light (laser). 2) Flow cytometry Thanks!