Comparison of conventional and modern techniques
• In conventional techniques, isolation and manipulation of DNA or RNA is not applied.
• Experiments are set based on phenotypic characters and the results are expressed based on observations or quantitative measurements.
• The basic materials of modern biotechnology are nucleic acids (NA) and proteins (P).
• NA and P are isolated from tissue, purified and subjected to certain processes.
• NAs are amplified in bacteria or by polymerase chain reaction (PCR), cut with restriction enzymes and hybridized with a different NA, If necessary, or run on an Agarose Gel Electrophoresis, stained and visualized.
• Likewise, proteins are purified, run on an SDS-PAGE gel, stained/immunoblotted and visualized.
Proof of transforming factor
In vivo Experiment
• In 1928, Frederick Griffith described a transforming principle that transmitted the ability of bacteria to cause pneumonia in mice.
“transforming principle.”
In vitro experiments
•Oswald Avery, Colin MacLeod, and Maclyn McCarty used this assay to show in 1944 that Griffith’s transforming principle was DNA. the transforming factor could be destroyed by enzymes that degrade DNA (deoxyribonucleases) but not by protease or ribonuclease enzymes.
Nucleic acid hybridization and PCR based methods
• GISH, Genomic in situ Hybridization
• FISH, Flourosence in situ Hybridization
• RFLP, Restrictrd Fragment Lenght Polimorphism
• SOUTHERN BLOTTING
• RAPD, Randomly Amplified Polimorphic DNA
• AFLP, Amplified Fragment Lenght Polimorphism
• VNTR, Variable Number of Tandem Repeatd
Gene Expression Analysis Methods
Analysis at the level of gene transcription:
RNA expression and localization (Transcription)
Northern blot
In situ hybridization
RNase protection assay (RPA)
Reverse transcription-PCR (RT-PCR)
Quantitative real time PCR (Q-PCR)
Analysis at the level of translation:
Protein expression and localization (Translation)
Western blot
In situ analysis
Enzyme-linked immunosorbent assay (ELISA)
Antisense technology (Inhibition)
Antisense oligonucleotides
RNA interference (RNAi)
Analysis of DNA–protein interactions
Electrophoretic mobility shift assay (EMSA)
DNase I footprinting
Chromatin immunoprecipitation (ChIP) assay
Analysis of protein–protein interactions
Pull-down assay
Yeast two-hybrid assay
Coimmunoprecipitation assay
Fluorescence resonance energy transfer (FRET)
Structural analysis of proteins
X-ray crystallography
Nuclear magnetic resonance (NMR) spectroscopy
Cryoelectron microscopy
Atomic force microscopy (AFM)
Application areas
• Taxonomical applications,
• Agricultural applications,
• Genomemaps,
• Identification of the individual genotype,
• Detection of DNA polymorphism,
• Detection of quantitative trait loci,
• Identification of parents and relatives,
• Diagnosis of diseases and genetic defects,
• Prenatal Diagnosis
• Forensic Medicine
HOMOGENIZATION is commonly defined as a chemical or physical treatment by which the composition or structure of a substance (solid, liquid or gas) or mixture of substances is made uniform.
EXTRACTION is a separation process consisting of the separation of a substance from a matrix.
PURIFICATION means the removal of unwanted impurities present in an organic compound.
The main steps of DNA extraction
1. Break cell wall and membrane
2. Make free the cell ingredient/content/subtances
3. Eliminate polysaccharides, polyphenols, lipids, proteins, etc. except for DNA
4. Precipitate DNA
5. Wash and Purify DNA 6. Keep in specific solvent in fridge for along time until use.
DNA Markers: DNA fragments generated by restriction endonuclease enzymes to compare and identify the size, specifity and some features of unknown DNA fragment are called DNA markers or genetic markers.
GENOME
•A genome is an organism’s complete set of genetic instructions (total genes).
•Each genome contains all of the information needed to build that organism and allow it to grow and develop.
• This code is determined by the order of the four nucleotide bases that make up DNA, adenine, cytosine, guanine and thymine, A, C, G and T for short.
DNA
• DNA or deoxyribonucleic acid is a long molecule that contains our unique genetic code.
• The two strands run in the opposite direction (antiparallel) to each other so that one runs 5’ to 3’ and one runs 3’ to 5’, they are called the sense strand and the antisense strand, respectively.
GENE
• Genes are small sections of DNA within the genome that code for proteins. They contain the instructions for our individual characteristics– like eye and hair colour.
