Genetic Analysis and Recombinant DNA Technology
Chapter 08 Genetic Analysis and Recombinant DNA Technology
Section 8.1: Tools and Techniques of Recombinant DNA Technology
Learning Outcomes:
Provide examples of practical applications of modern genetic technologies.
Explain the role of restriction endonucleases in the process of recombinant DNA technologies.
List the steps in the polymerase chain reaction (PCR) and discuss one disadvantage to this technique.
Describe how recombinant DNA is created and discuss its role in gene cloning.
Applications of Recombinant DNA Technology
From Basic Science:
Utilizing DNA to identify individuals.
Fixing underlying genetic mutations to treat diseases.
Using CRISPR technology to repair genetic mutations in a way that the fixes can be transferred to future generations.
DNA: The Raw Material
Intrinsic Properties of DNA:
DNA strands separate when exposed to temperatures just below boiling.
When separated, the nucleotides are exposed allowing for identification, replication, and transcription of the DNA molecule.
Complementary nucleotides will hydrogen bond to each other, and the strands will regain their double-stranded form upon slow cooling.
Heating and Cooling of DNA:
Heating causes DNA to denature (lose its hydrogen bonding) and separate into two strands.
When cooled, the two strands rejoin at complementary regions; they do not have to originate from the same organism as long as they have matching nucleotides.
Restriction Endonucleases
Function: Enzymes that clip DNA crosswise at selected locations.
Recognize foreign DNA to act upon.
Break phosphodiester bonds between adjacent nucleotides on both strands of DNA.
Each endonuclease recognizes sequences of 4 to 10 base pairs.
Protect bacteria and archaea against incompatible DNA from bacteriophages or plasmids.
Palindromes and Sticky Ends
Palindromic Sequences:
Sequences of DNA that are identical when read from the 5' to 3' direction on one strand and the 5' to 3' on the other strand.
Sticky Ends:
Staggered symmetrical cuts that leave short tails of 4 to 5 bases on each strand.
Base pair with complementary tails on other DNA fragments or plasmids.
Restriction Fragments and RFLPs
Restriction Fragments:
Pieces of DNA produced by restriction endonucleases.
Restriction Fragment Length Polymorphisms (RFLPs):
Differences in the cutting patterns of specific endonucleases resulting in restriction patterns of different lengths.
Allow direct comparison of DNA of two different organisms at specific sites.
Ligase and Reverse Transcriptase
Ligase:
Seals sticky ends together by rejoining the phosphate-sugar bonds cut by endonucleases.
Main application is the final splicing of genes into plasmids and chromosomes.
Reverse Transcriptase:
Enzyme that replicates HIV and other retroviruses, converting RNA into DNA.
Making cDNA From Eukaryotic mRNA
Complementary DNA (cDNA):
Made from messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and others.
Useful in synthesizing eukaryotic genes from mRNA transcripts, which are free of introns.
CRISPR Technology
CRISPR - Clustered Regularly Interspaced Short Palindromic Repeats:
Short lengths of DNA with repeating nucleotides found in bacteria and archaea.
Enzyme Cas9 cuts out foreign DNA left behind by invading bacteriophages or plasmids.
Scientists exploit this to cut DNA in virtually any organism precisely.
Polymerase Chain Reaction (PCR)
Function:
Rapidly increases the amount of DNA in a sample without the need for cultures or complex purification techniques.
Can replicate a target DNA from a few copies to billions in a few hours.
Sensitivity:
Can detect cancer from a single cell and diagnose infections from a single gene copy.
Steps of DNA Replication in PCR
Uses events of DNA replication:
Opening of the double helix.
Using exposed strands as templates.
Addition of primers.
Action of DNA polymerase.
Specialized Ingredients Used in PCR
Primers:
Synthetic oligonucleotides of a known sequence (15-30 bases) that indicate the start of DNA amplification.
DNA Polymerases:
Enzymes responsible for DNA replication.
High temperatures necessitate the use of DNA polymerases isolated from thermophilic bacteria like Taq polymerase from Thermus aquaticus and Vent polymerase from Thermococcus litoralis.
PCR Technique
Utilizes a thermal cycler performing cyclic temperature changes.
Three Basic Steps:
Denaturation.
Priming.
Extension.
Cyclic repetition amplifies DNA exponentially (e.g., 68 billion molecules from one copy).
Additional PCR Techniques
Real-time PCR:
Detect products during the reaction instead of at endpoint.
Adaptation to Analyze RNA:
Reverse transcriptase converts RNA to cDNA which can be amplified with PCR.
Loop-mediated Isothermal Amplification (LAMP):
Does not go through temperature variations to complete PCR.
Methods in Recombinant DNA Technology
Definition:
Deliberately removing genetic material from one organism and combining it with another to form genetic clones.
Cloning Process:
Removal of a gene from a genetic donor (animal, plant, or microorganism).
Insertion of the gene into a vector (plasmid or virus) that will be inserted into a cloning host.
Translation of the gene into its corresponding protein product.
Strategies for Obtaining Genes in Isolated State
Common Strategies:
Removal of DNA from cells using endonucleases to separate into fragments.
Synthesis from isolated mRNA transcripts using reverse transcriptase (to create cDNA).
Amplification of the gene using PCR.
Genomic Libraries
Definition:
Genes maintained in a cloning host and vector, similar to microbial pure culture.
Collections of cDNA clones representing the entire genome of several organisms.
Cloning Vectors
Plasmids:
Small, well-characterized, easy to manipulate, can be transferred into host cells through transformation, carry genetic markers for antibiotic resistance.
Bacteriophage:
Naturally capable of injecting DNA into bacterial hosts via transduction.
Important Aspects of Cloning Vectors
Origin of Replication (ORI):
Must be replicated by the DNA polymerase of the cloning host.
Size Acceptance:
Must accept DNA of the desired size.
Drug Resistance Gene:
Typically contain a gene that provides drug resistance to their cloning host.
Desirable Features in a Microbial Cloning Host
Characteristics:
Rapid turnover and fast growth rate.
Large-scale growth using standard culture methods.
Nonpathogenic.
In-depth genome mapping.
Capacity to accept plasmid or bacteriophage vectors.
Maintain foreign genes through multiple generations.
High yield of proteins from expressed foreign genes.
Example of Recombinant DNA Technology
Production of Alpha-2a Interferon (Roferon-A):
Used to treat hairy cell leukemia and Kaposi’s sarcoma.
Human alpha interferon gene codes for a polypeptide and is isolated from mRNA transcripts free of introns.
Ingredients for Gene Cloning
Essential Components:
The gene of interest (e.g., interferon) and a cloning vector (usually a plasmid).
First Steps in Gene Cloning
Procedure:
Digest the donor gene and plasmid using the same restriction enzyme, producing complementary sticky ends.
Base-pair the gene and plasmid together; ligase seals the bonds covalently.
Introduce the recombinant plasmid through transformation into the cloning host (lab strain of E. coli).
Use selective medium to identify recombinant clones.
Synthesis and Purification of Protein in Gene Cloning
As the cells multiply, each cell carries the plasmid along with its chromosome.
Once cloned and tested, the recombinant strain cultures without redoing the cloning step.
The donor gene is transcribed to mRNA and translated into the protein product.
Synthetic Biology
Definition:
Creation of new biological molecules and organisms from scratch.
In 2010, scientists created a self-replicating bacterium from DNA nucleotides.
Applications: Precise chemical synthesis to replace compounds missing in diseases, customized immune components, and targeting cancerous cells or pathogens.
Section 8.2: DNA Analysis
Learning Outcomes:
Describe how gel electrophoresis is used for DNA analysis.
Outline steps in DNA profiling.
Summarize the DNA sequencing process.
Discuss the significance of single nucleotide polymorphisms (SNPs) in DNA analysis.
Describe uses of microarray technology.
Genome Analysis: Maps and Profiles
Analysis of DNA reveals genetic abnormalities, ancestry, and determines if samples are the same.
Example: Identifying a criminal via DNA from a hair at a crime scene.
Gel Electrophoresis
Function:
Produces readable patterns of DNA fragments from samples subjected to an electric current in agar gel.
DNA, negatively charged from phosphate groups, moves toward the positive pole; speed depends on fragment size.
Staining allows for visualization of DNA fragment positions.
Distinctive Electrophoresis Patterns
Useful in characterizing DNA fragments and comparing genetic similarities across samples.
Nucleic Acid Hybridization and Probes
Hybridization:
Different nucleic acids unite at complementary regions; ssDNA can hybridize with other ssDNA or RNA.
Gene Probes
Definition:
Short DNA stretches of known sequences that base pair with complementary sequences in a sample.
Carry reporter molecules like fluorescent dyes for visualization of hybridization areas.
Uses of Gene Probes
Useful for diagnosing infections and identifying unknown bacterial or viral cultures.
Nucleic Acid Hybridization Test
Procedure that doesn’t require electrophoresis; DNA is isolated, denatured, and combined with specific probes for hybridization, subsequently developed and observed for hybridization areas.
Fluorescent In Situ Hybridization (FISH)
Probes applied to intact cells and microscopically observed locate specific genetic markers on genes.
Useful for identifying genes on chromosomes and bacteria without culturing.
Also detects RNA in cells/tissues.
Size of DNA Analysis
Relative sizes of nucleic acids denoted by base pairs (bp):
Palindromic sequences: 4 to 10 bp.
Average gene in E. coli: 1,300 bp or 1.3 kb.
Entire E. coli genome: 4,700,000 bp or 4.7 Mb.
Human mitochondrion: 16 kb; Epstein–Barr virus: 172 kb; Human genome: 6 billion bp (46 chromosomes).
DNA Profiles: A Unique Picture of the Genome
DNA Profiling:
Forensic tool relying on small unique differences in DNA between individuals (same species).
Restriction enzymes cut DNA, producing fragments of varying lengths in digested samples from different individuals.
Restriction Fragment Length Polymorphism (RFLP)
Function:
Restriction enzymes cut DNA at specific sites; whether the strand has the site determines if it is cut into pieces.
DNA Fingerprinting Dependence on RFLP
Involves digesting samples with restriction enzymes, separating using gel electrophoresis, leading to distinct banding patterns that depend on variations in DNA.
Sequence Maps
Function:
Detailed genome maps indicating base order in plasmids, chromosomes, or genomes.
Thousands of organisms sequenced; genetic similarities noted across species (e.g., humans share 80% DNA with mice).
Whole-Genome Shotgun Sequencing: Steps 1–4
Process involves breaking down whole genomes into smaller fragments, purifying, transforming into E. coli, sequencing clonal inserts, and assembling contigs.
Whole-Genome Shotgun Sequencing: Steps 5–7
Utilize computer programs to align sequences and construct larger contiguous sets (contigs), followed by human analysis for irregularities and ambiguities.
High-Throughput Genome Sequencing
Development of sophisticated systems for quicker genome sequencing with minimal human intervention.
Cost to Sequence a Human Genome (2001–2020)
Initially took 13 years & $3 billion; now can be completed in an afternoon at less than $1,000.
Genomics and Bioinformatics
Managing data from high-throughput sequencing, analyzing and classifying genes, determining protein sequences, and gene functions.
Annotating the Genome
Determines functional information of gene sequences, aiding in understanding cell function, disease, development, and aging.
Single Nucleotide Polymorphism (SNP)
Definition:
SNPs involve single nucleotide alterations, impacting individual traits, with 10 million SNPs mapped in the human genome; used to identify disease risks (e.g., thrombophilia from a mutation in the clotting factor V gene).
Example of SNP Associated with Disease
Normal vs. Excessive Clotting:
Normal: Codon GCT to CGA transcribes to Arginine (Arg), allowing proper clotting.
Excessive: Codon GTT to CAA changes Arginine to Glutamine (Gln), preventing inhibition and causing excessive clotting.
DNA Microarray Analysis
Allows biologists to monitor gene expression levels across thousands of genes in cells.
Uses of Microarrays
Developing diagnostic tests that target specific patterns of gene expression to identify cancer subtypes.
Section 8.3: Genetic Approaches to Healing Disease
Learning Outcomes:
Define personalized medicine.
Provide examples of recombinant products contributing to human health.
List recombinant bacteria and plants with purpose.
Differentiate somatic and germline gene therapy.
Describe the impact of miRNAs on human diseases.
Explain CRISPR-Cas9 usage for curing genetic diseases.
Products of Recombinant DNA Technology
Uses:
Producing recombinant organisms and protein products, nucleotide sequences, and medications that can’t be manufactured otherwise.
Large-scale hormone and enzyme manufacture like insulin & human growth hormone.
Current Medicines from Recombinant DNA Technology
Immune Treatments:
Interferons for cancer, MS, viral infections.
Interleukins for white blood cell regulation.
TNF for cancer treatment.
Biologics like Remicade® and Humira® for autoimmune disorders.
Hormonal Treatments:
Erythropoietin (EPO) for anemia; Human Growth Hormone (HGH) for growth issues; rhDNase for cystic fibrosis; tPA for dissolving blood clots; PEG-SOD to minimize brain and organ damage.
Vaccines and Additional Medicines
Vaccines for hepatitis B, HPV, Haemophilus influenzae type b; Factor VIII for hemophilia A.
Genetically Modified Organisms (GMOs)
Definition:
Transgenic organisms produced by integrating foreign genes to create recombinant microbes, plants, and animals.
Uses of Recombinant Microbes
- Modifying Pseudomonas syringae to prevent ice crystals; Pseudomonas fluorescens to destroy insects; making plants resistant to pests by inserting insecticide genes from Bacillus thuringiensis.
Controversy Surrounding Transgenic Plants
Concerns about gene sharing leading to “superweeds” versus the fact that genes swap in nature.
Inserting insect-resistance genes can decrease the need for pesticides.
Gene Therapy
Goal:
Repair or correct faulty genes for permanent cures of diseases.
Strategies for Gene Therapy
Cloning normal genes in retrovirus/adenovirus vectors, infecting patient tissues, reintroducing transfected cells, or using naked DNA for direct introduction into tissues.
Gene Therapy Treatments (As of 2022)
Approved for 13 diseases: e.g., spinal muscular atrophy, inherited vision loss, and acute lymphoblastic leukemia.
Somatic Cell Gene Therapy:
Changes are permanent in the individual but not heritable.
Germline Therapy:
Genes inserted into egg/sperm/embryos; changes are heritable.
Small RNAs as Medicine
miRNAs silence gene expression; introducing them can inhibit cancer growth in mice (e.g., lung and liver cancers).
Inhibiting miRNAs:
Stopping specific miRNAs can halt ovarian and breast cancers; lowering cholesterol; inhibit hepatitis C virus multiplication in chimpanzees.
Tests in Humans
The first successful human application of interfering RNAs involved nasal sprays with miRNA, showing reduced RSV infections in volunteers.
CAR-T Cell Treatment of Cancer
DNA recombination techniques modify T cells to battle cancer cells.
Patients’ T cells are altered to recognize and destroy cancer cells; currently an expensive treatment.
CRISPR-Cas9 System
A naturally occurring gene-editing system in bacteria; awarded the 2020 Nobel Prize in Chemistry for its precise gene editing capabilities.
Awardees: Dr. Jennifer Doudna and Dr. Emmanuelle Charpentier.
Concept Checks
Section 8.1:
Describe two scenarios for manipulating genetic information for human benefit.
DNA can be cut by enzymes called ____.
cDNA is produced using the enzyme ____.
True/False: PCR uses specialized DNA polymerase adapted to high temperatures.
List two cloning vectors and two cloning hosts.
Section 8.2:
Outline steps in whole-genome shotgun sequencing.
____ genomes provide complete understanding of cell function and disease.
DNA profiling depends on ____ enzymes.
A(n) ____ allows scientists to view gene expression in cells.
Section 8.3:
List three products from recombinant DNA technology.
Recombinant organisms with foreign genes are called ____.
____ therapy refers to gene therapy in eggs, sperm, or embryo.
True/False: Naked DNA is often used in gene therapy.
True/False: CRISPR-Cas9 naturally occurs in bacteria.