Chp 8 PPT

Chapter 08: Genetic Analysis and Recombinant DNA Technology

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.

Definition: Combination of genetic material from different organisms.
Applications:
- Crime scene investigation.
- Genetic disease treatments through mutation correction.
- CRISPR technology for permanent genetic alterations.

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:
- DNA separates into strands when denatured.
Cooling:
- Strands rejoin at complementary sequences, regardless of organism origin.

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.

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:
- Pieces of DNA resulting from restriction enzyme cuts.
RFLPs (Restriction Fragment Length Polymorphisms):
- Variation in DNA fragment lengths due to different restriction patterns.

Ligase:
- Seals DNA fragments by rejoining phosphate-sugar bonds.
- Finalizes gene splicing into vectors.
Reverse Transcriptase:
- Converts RNA into DNA, used in retroviral replication.

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.

Overview:
- Amplifies DNA samples rapidly.
Steps:
- Denaturation, primers addition, extension by DNA polymerase.
Specialized Requirements:
- Uses thermophilic DNA polymerases due to high-temperature cycling.

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.

Characteristics for effective microorganism hosts:
- Fast growth, nonpathogenic, well-mapped genomes, high yield of expressed proteins.

Alpha-2a Interferon:
- Used for treating hairy cell leukemia and Kaposi’s sarcoma. Derived from a human gene processed from mRNA.

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.

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.

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.

Function: Monitors gene expression across thousands of genes simultaneously.
Applications: Design of diagnostic tests to identify specific diseases based on gene expression patterns.