Slide 1

Biotechnology- Principles and Processes

• Introduction to biotechnology
• Importance of biotechnology in various fields
• Overview of biotechnology processes

Slide 2

Polymerase Chain Reaction (PCR)

• Definition and purpose of PCR
• Steps involved in PCR:
  • Denaturation
  • Annealing
  • Extension
• Role of DNA polymerase

Slide 3

Denaturation

• Heat-induced separation of DNA strands
• Temperature and duration of denaturation vary depending on the DNA template
• DNA strands become single-stranded

Slide 4

Annealing

• Temperature is lowered to allow primers to bind to the DNA template
• Primers are short DNA sequences that are complementary to the target DNA region

Slide 5

Extension

• Temperature is increased to allow DNA polymerase to attach nucleotides to the primer
• DNA synthesis occurs from the 3’ end of the primer in the 5’ to 3’ direction
• Extension continues until the desired DNA region is amplified

Slide 6

Applications of PCR

• Amplification and cloning of DNA
• Genetic testing and diagnosis
• Forensic analysis
• Disease detection and monitoring
• Environmental studies

Slide 7

Real-Time PCR

• Utilizes fluorescent dyes or probes to monitor DNA amplification in real-time
• Allows for quantification of DNA during the PCR process
• Different types of detection systems used in real-time PCR

Slide 8

Reverse Transcription PCR (RT-PCR)

• Converts RNA into complementary DNA (cDNA) before PCR amplification
• Useful for studying gene expression and identifying gene mutations
• Steps involved in RT-PCR

Slide 9

Multiplex PCR

• Amplifies multiple target DNA regions simultaneously using multiple primers in a single reaction
• Provides efficient and rapid analysis of multiple samples
• Applications of multiplex PCR in genetic testing and diagnostics

Slide 10

Limitations and Troubleshooting of PCR

• Contamination issues
• Primer design considerations
• Optimization of PCR conditions
• Troubleshooting common problems (e.g., non-specific amplification, low yield)

======= 11.

Gel Electrophoresis

• Technique used to separate DNA fragments by size
• Principle of gel electrophoresis
• Agarose gel vs. polyacrylamide gel
• Loading samples onto the gel
• Application of an electric current to drive DNA migration

DNA Analysis and Visualization

• Staining DNA in the gel
• UV light visualization
• Ethidium bromide and other DNA stains
• DNA ladder for size comparison
• Documentation of gel images

DNA Sequencing

• Definition and importance of DNA sequencing
• Sanger sequencing method
• DNA sequencing by synthesis (Next-Generation Sequencing)
• Applications of DNA sequencing in various fields (genomics, medicine, evolutionary biology)

Recombinant DNA Technology

• Definition and purpose of recombinant DNA technology
• Techniques involved in recombinant DNA technology:
  • Restriction enzymes
  • DNA ligation
  • Transformation
• Cloning vectors and their types (plasmids, viral vectors)

Restriction Enzymes

• Definition and role of restriction enzymes
• Classification of restriction enzymes
• Recognition sequence and cutting pattern
• Applications of restriction enzymes in biotechnology

DNA Ligase

• Definition and role of DNA ligase
• Sealing of DNA fragments using DNA ligase
• Role of DNA ligase in DNA replication and repair
• Applications of DNA ligase in recombinant DNA technology

Transformation

• Definition and process of transformation in DNA technology
• Techniques for achieving transformation:
  • Chemical transformation
  • Electroporation
  • Microinjection
• Factors influencing transformation efficiency

Cloning Vectors

• Definition and purpose of cloning vectors
• Types of cloning vectors:
  • Plasmids
  • Phages
  • Cosmids
  • Bacterial artificial chromosomes (BACs)
• Features required for a successful cloning vector

Genomic Library Construction

• Purpose of genomic library
• Construction of a genomic library:
  • Isolation and fragmentation of genomic DNA
  • Cloning fragments into a suitable vector
  • Introduction of recombinant vectors into host cells

Applications of Recombinant DNA Technology

• Production of recombinant proteins:
  • Insulin
  • Human growth hormone
  • Enzymes
• Gene therapy
• Genetic engineering of crops
• DNA fingerprinting and forensic analysis
• Transgenic animals

Biotechnology- Principles and Processes - Polymerase Chain Reaction Stages

• Denaturation
• Annealing
• Extension

Denaturation

• Heat-induced separation of DNA strands
• Temperature and duration vary based on the DNA template
• DNA strands become single-stranded

Annealing

• Temperature is lowered to allow primers to bind to the DNA template
• Primers are short DNA sequences complementary to the target region
• Ensures specificity of amplification

Extension

• Temperature is increased for DNA polymerase activity
• DNA synthesis occurs from the 3’ end of primers
• Extension continues until the desired DNA region is amplified

Applications of PCR

• DNA amplification and cloning
• Genetic testing and diagnosis
• Forensic analysis
• Disease detection and monitoring
• Environmental studies

DNA Amplification and Cloning

• PCR allows amplification of a specific DNA fragment
• Useful for producing large amounts of DNA for further analysis or manipulation
• Can be used for gene cloning and recombinant DNA technology

Genetic Testing and Diagnosis

• PCR-based tests can detect specific genetic or pathogenic sequences
• Used for prenatal testing, carrier screening, and disease diagnosis
• Enables early detection and personalized medicine

Forensic Analysis

• PCR amplification allows efficient analysis of small DNA samples
• Used in criminal investigations, paternity testing, and identification of human remains
• High sensitivity and specificity aid in accurate DNA profiling

Disease Detection and Monitoring

• PCR can detect genetic mutations associated with diseases
• Enables monitoring of treatment response and disease progression
• Used in infectious disease diagnostics (e.g., detection of viral or bacterial DNA)

Environmental Studies

• PCR-based techniques help identify and study microorganisms in environmental samples
• Used in water and soil quality assessment, microbial ecology, and biodiversity studies
• Enables monitoring of environmental pollutants and pathogens