Chp 8 PPT

Chapter 08: Genetic Analysis and Recombinant DNA Technology

Section 10.1: Tools and Techniques of Genetic Engineering

• Learning Outcomes:

  • Practical applications of modern genetic technologies:

    • Identifying a suspect in crime cases.

    • Correcting genetic mutations to treat diseases.

    • Using CRISPR for genetic repair that can be inherited.

  • Role of restriction endonucleases in genetic engineering.

  • Function of gel electrophoresis for DNA analysis.

  • Polymerase chain reaction (PCR) steps and its disadvantages.

  • Creation of recombinant DNA and its importance in gene cloning.

Recombinant DNA Technology

• Definition: Combination of genetic material from different organisms.

• Applications:

  • Crime scene investigation.

  • Genetic disease treatments through mutation correction.

  • CRISPR technology for permanent genetic alterations.

DNA: The Raw Material

• Intrinsic Properties:

  • Helicase unwinds DNA in lab and cellular environments.

  • DNA denatures at high temperatures and reassociates when cooled.

  • Nucleotides become accessible for identification and replication.

Heating and Cooling of DNA

• Heating:

  • DNA separates into strands when denatured.

• Cooling:

  • Strands rejoin at complementary sequences, regardless of organism origin.

Restriction Endonucleases

• Functionality:

  • Enzymes that cut DNA at specific positions.

  • Break phosphodiester bonds between nucleotides.

  • Recognize sequences of 4 to 10 base pairs.

• Protection Mechanism:

  • Used by bacteria to defend against foreign DNA.

Palindromes and Sticky Ends

• Palindrome Recognition:

  • Clipping sequences that are identical when read in both directions.

• Sticky Ends:

  • Staggered cuts that create tails on the fragments, allowing for base-pairing with other DNA.

Restriction Fragments and RFLPs

• Restriction Fragments:

  • Pieces of DNA resulting from restriction enzyme cuts.

• RFLPs (Restriction Fragment Length Polymorphisms):

  • Variation in DNA fragment lengths due to different restriction patterns.

Ligase and Reverse Transcriptase

• Ligase:

  • Seals DNA fragments by rejoining phosphate-sugar bonds.

  • Finalizes gene splicing into vectors.

• Reverse Transcriptase:

  • Converts RNA into DNA, used in retroviral replication.

CRISPR Technology

• Definition: Short DNA sequences with repeating nucleotides from bacteria.

• Cas9 Enzyme:

  • Recognizes and cuts foreign DNA, utilized by scientists for targeted DNA editing in various organisms.

Applications of PCR (Polymerase Chain Reaction)

• Overview:

  • Amplifies DNA samples rapidly.

• Steps:

  • Denaturation, primers addition, extension by DNA polymerase.

• Specialized Requirements:

  • Uses thermophilic DNA polymerases due to high-temperature cycling.

Gene Cloning Process

• Steps:

  • Isolate the gene of interest, typically through digesting DNA with restriction enzymes.

  • Insert gene into vectors (plasmids or viruses) for replication in host organisms.

• Cloning Vectors:

  • Plasmids and bacteriophages used to carry and propagate genes.

Desirable Features of Cloning Hosts

• Characteristics for effective microorganism hosts:

  • Fast growth, nonpathogenic, well-mapped genomes, high yield of expressed proteins.

Example of Recombinant DNA Technology

• Alpha-2a Interferon:

  • Used for treating hairy cell leukemia and Kaposi’s sarcoma. Derived from a human gene processed from mRNA.

Synthetic Biology and Gene Therapy

• Synthetic Biology: Creation of new biological entities; example includes self-replicating bacteria.

• Gene Therapy: Corrects faulty genes to cure diseases via vectors, such as retroviruses.

Whole-Genome Shotgun Sequencing

• Method:

  • Genome fragmenting, electrophoresis, cloning, and sequencing using automated techniques.

• Cost Reduction:

  • Significant decrease in time and cost of sequencing human genomes from $3 billion over 13 years to approximately $1,000 today.

Single Nucleotide Polymorphisms (SNPs)

• Definition: Variations at single nucleotide positions affecting individual traits, with millions mapped in the human genome.

• Implications: Some SNPs are linked to increased disease risk, exemplified by thrombophilia.

DNA Microarray Technology

• Function: Monitors gene expression across thousands of genes simultaneously.

• Applications: Design of diagnostic tests to identify specific diseases based on gene expression patterns.