Lecture 33 - DNA Techniques

Objectives of DNA Techniques

  • Define roles of Nucleases, Restriction Endonucleases, and DNA Ligases in DNA cloning.

  • Understand plasmids and vectors in DNA cloning.

  • Learn vocabulary: Transformation, recombinant, SNP, etc.

  • Comprehend Polymerase Chain Reaction (PCR) process and its applications.

  • Discuss DNA Sequencing and Microarray technologies.

Nucleases

  • Role: Hydrolytically cleave nucleic acids.

    • Types:

    • DNases (deoxyribonuclease): Cleaves DNA.

    • RNases (ribonuclease): Cleaves RNA.

  • Types of Activity:

    • Endonucleases: Cleave internal bonds, producing fragments.

    • Exonuclease: Cleave ends of DNA/RNA, can work from either end (5'-to-3' or 3'-to-5').

Restriction Endonucleases

  • Function: Recognize palindromic DNA sequences (4-8 bp), called restriction sites.

  • Outcome: Resulting pieces are restriction fragments, cleaving double-stranded DNA to create sticky or blunt ends.

  • Sticky Ends: Easier to clone due to ability to base pair directionally.

    • Example: EcoRI, HindIII, BamHI

  • bacteriophage - bacteria have a host protection,which can be exploited by using bacteriophages for gene transfer, allowing scientists to introduce foreign DNA into bacterial cells effectively.

Agarose Gel Electrophoresis

  • Purpose: Separate DNA fragments by size.

  • Process:

    • DNA fragments negatively charged, migrate towards anode.

    • Small fragments (~10-40 bp) analyzed in polyacrylamide gels; larger fragments (~50-50,000 bp) in Agarose gels.

    • Gel Composition: Higher percentage agarose (like 2%) is used for smaller fragments; lower (0.5%) for larger.

  • Visualization: Use of a tracking dye and ethidium bromide under UV light to see bands on a gel.

  • Ethidium bromide intercalates between the bases of DNA, allowing for the detection of nucleic acids when visualized under UV light.

Cloning Techniques in Forensics

  • RFLP (Restriction Fragment Length Polymorphism): Requires larger amounts of DNA, not degraded.

  • PCR (Polymerase Chain Reaction): Requires smaller amounts, can amplify from degraded samples.

    • STR (Short Tandem Repeat) Analysis: Utilizes PCR to analyze specific regions of DNA that are highly variable among individuals, making it ideal for forensic identification.

    • Mitochondrial DNA Analysis: Focuses on non-nuclear DNA inherited maternally, useful for identifying remains when nuclear DNA is unavailable.

  • Application of RFLP: Distinguishing disease genes via unique restriction patterns (ex., MboI).

Southern Blotting

  • Purpose: Detect specific DNA sequences in a pool of unspecific DNA.

  • Creating a DNA Fingerprint: Based on RFLPs, facilitating forensic analysis, paternity testing and ancient DNA analysis.

  • DNA Fingerprinting - A method that utilizes variations in the number of repeats of specific DNA sequences to distinguish individuals, which can be critical in criminal investigations and historical genetics.

    • Applications: Used in various fields such as medicine, anthropology, and genealogy, providing insights into genetic diversity and lineage.

    • Limitations: While powerful, DNA fingerprinting may face challenges such as the potential for sample degradation, contamination, and statistical misinterpretation, necessitating careful handling and analysis protocols.

Recombinant DNA Technology

  • Purpose: Isolate and study genes; accomplished by splicing DNA of interest into cloning vectors (like plasmids).

  • Steps:

    1. Restriction enzyme cuts DNA at specific sequence.

    2. Mix with DNA from other sources to ensure cohesive ends can base-pair.

    3. Use DNA ligase to bond DNA backbones.

Transformation of Bacteria

  • Definition: Process of introducing foreign DNA into bacteria.

    • Methods:

    • Heat-shock transformation: Using calcium chloride, cold shock then heat to introduce plasmids. Electroporation: Applying an electric field to increase cell membrane permeability, allowing plasmids to enter.

    • Electroporation: High voltage pulse creates pores in cell wall. Lipofection: Utilizes lipid-based reagents to facilitate the uptake of DNA by fusing with the bacterial cell membrane.

Plasmids - vehicles for cloning that carry genes of interest, allowing for gene manipulation and expression in host cells.

  • Viral Vectors: Modified viruses that can deliver genetic material into host cells efficiently, often used in gene therapy.

  • CRISPR/Cas9: A revolutionary gene-editing tool that allows for precise alterations to DNA sequences.

Plasmid DNA - they can not carry larger sizes of DNA well.

For larger DNA fragments, alternative methods such as BACs (Bacterial Artificial Chromosomes) or YACs (Yeast Artificial Chromosomes) are utilized, providing a more suitable framework for the manipulation of extensive genetic sequences.

Inserting a Gene into a Vector

1) The gene of interest is selected based on the desired trait or function, and it is then amplified using PCR techniques to ensure sufficient quantity for insertion.

2) The next step involves the digestion of both the vector and the gene using specific restriction enzymes, creating compatible ends for ligation.

3) The ligation process follows, where the gene is inserted into the vector using DNA ligase, sealing the sugar-phosphate backbone and forming a recombinant DNA molecule.

Selection of recombinant DNA in Bacteria

Selection - is a process designed to facilitate the identification of recombinant bacteria that prevents the growth of non-transformed bacteria and bacteria that contain plasmids without foreign (recombinant) DNA

Antibiotic selection - plate transformed bacterial cells on plates containing different antibiotics to identify recombinant bacteria and non-transformed bacteria.

  • first human protein expressed via recombinant techniques was Insulin and next was Growth hormone.

PCR (Polymerase Chain Reaction)

  • Developed by: Kary Mullis in the 1980s.

  • Process: Amplifies specific DNA sequences via cycles of denaturation, annealing, and extension.

  • Stages:

    1. Denaturation (~94-96°C)

    2. Annealing (55-65°C)

    3. Extension (70-75°C)

  • Taq Polymerase: Optimal for high-temperature applications.

Applications of PCR

  • Forensics: Amplifying DNA from samples.

  • Diagnosis: Genetic conditions and pathogen detection based on small sample sizes.

  • Gene Cloning: PCR for producing DNA fragments for cloning vectors.

  • quantitative pcr - a technique used to amplify and simultaneously quantify a targeted DNA molecule, allowing for precise measurement of DNA concentrations in various applications such as gene expression analysis.

DNA Sequencing

  • Sanger Sequencing: Utilizes ddNTPs to terminate DNA strands at various lengths for sequence determination.

  • Modern Sequencing: Fast sequencing of DNA using fluorescently labeled dideoxynucleotides and capillary gels.

Single Nucleotide Polymorphisms (SNPs)

  • Definition: One nucleotide change in DNA sequences; significant for understanding genetic disorders (e.g., sickle cell anemia).

  • Application: Helps tailor treatments in precision medicine, especially in diseases like breast cancer with SNP identification in BRCA genes.

Gene Microarrays

  • Function: Analyze gene expression and identify SNPs using DNA fragments on slides compared against patient DNA.

  • Marker for Disease: Changes in gene expression can lead to new treatments.

Review of Techniques

  • Nucleases

  • Restriction Digests

  • RFLP and DNA Fingerprinting

  • DNA Cloning via Transformation

  • PCR and its multiple applications

  • DNA Sequencing and Microarray Technologies

Questions?