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Chapter 20
1. Gene Cloning Using Vectors
What is Gene Cloning?
Gene cloning is the process of isolating and making many copies of a specific gene using vectors.
Uses of Gene Cloning
● DNA sequencing ● DNA probes ● Protein expression (e.g., insulin production)
Key DNA Types in Cloning
Chromosomal DNA
● Source of the gene of interest
Vector DNA
● Carrier DNA that delivers the gene into a host cell ● Can replicate independently
Types of Vectors
Plasmids
● Circular DNA found in bacteria ● Contain: ○ Origin of replication ○ Antibiotic resistance genes
Viruses
● Infect cells and insert DNA
Restriction Enzymes
● Cut DNA at specific sequences (recognition sites) ● Recognition sequences are usually palindromic
Example: 5′ GAATTC 3′ 3′ CTTAAG 5′
Types of cuts:
● Sticky ends: single-stranded overhangs that can base-pair ● Blunt ends: straight cuts
DNA ligase joins DNA fragments together.
Steps of Gene Cloning
- Cut vector DNA and chromosomal DNA with the same restriction enzyme
- Mix DNA fragments so sticky ends base-pair
- Use DNA ligase to form recombinant DNA
- Insert DNA into bacteria (transformation)
- Plate bacteria on antibiotic-containing media
Blue-White Screening (lacZ gene)
● lacZ produces β-galactosidase
Blue colonies:
● lacZ functional ● No DNA insert
White colonies:
● lacZ disrupted ● DNA insert present
White colonies contain recombinant DNA.
Amplification of DNA
● Plasmids replicate within cells ● Bacteria divide rapidly ● Results in millions of copies of the gene
2. cDNA and DNA Libraries
cDNA (Complementary DNA)
● Primers (short DNA sequences) ● dNTPs (building blocks) ● Taq polymerase (heat-stable enzyme)
Three Steps of PCR
- Denaturation (~95°C) DNA strands separate
- Annealing (~50–65°C) Primers bind to DNA
- Extension (~72°C) DNA polymerase synthesizes new DNA
Amplification
● DNA doubles each cycle ● 20–30 cycles produce millions to billions of copies
Types of PCR
RT-PCR (Reverse Transcriptase PCR)
● Converts RNA to cDNA ● Used to measure gene expression
qPCR (Quantitative PCR)
● Measures DNA amount in real time using fluorescence
Key concept:
● Ct (Cycle Threshold) ○ Lower Ct = more starting DNA
5. DNA Sequencing (Sanger Method)
Key Idea
DNA replication is interrupted using special nucleotides.
Dideoxynucleotides (ddNTPs)
● Lack a 3′ OH group
● Stop DNA chain elongation
Process
- DNA is copied
- ddNTPs randomly terminate strands
- Fragments of different lengths form
- Fragments are separated by gel electrophoresis
- Sequence is determined
Modern Method
● Each base is labeled with a different fluorescent color ● Automated detection reads sequence
6. CRISPR-Cas Gene Editing
Definition
Gene editing is the intentional modification of DNA sequences.
Components
● sgRNA (guide RNA): targets specific DNA sequence ● Cas9: enzyme that cuts DNA
Mechanism
- sgRNA binds to target DNA
- Cas9 creates a double-strand break
- Cell repairs DNA
Repair Pathways
Nonhomologous End Joining (NHEJ)
● Error-prone ● Causes mutations or gene inactivation
Homology-Directed Repair (HDR)
● Uses template DNA ● Precise editing
1. Introduction to Biotechnology
Biotechnology refers to the use of living organisms or their products to benefit humans.
● Has existed for thousands of years (e.g., domestication, fermentation) ● Modern biotechnology uses molecular genetics (recombinant DNA)
Key Terms:
● Genetically modified organism (GMO): organism with altered DNA ● Transgenic organism: contains DNA from another species ● Transgene: gene transferred between species
2. Uses of Microorganisms in Biotechnology
Microorganisms are widely used in:
● Medicine production (e.g., insulin, antibiotics) ● Food production (cheese, yogurt, beer) ● Biological control of pests ● Bioremediation (pollution cleanup)
Recombinant Microorganisms in Medicine
Example: Insulin production
Insulin:
● Hormone that regulates glucose uptake ● Produced by pancreatic β cells
Problem:
● Diabetics cannot produce enough insulin
Solution using bacteria:
- Insert human insulin gene into bacterial plasmid
- Transform bacteria (usually E. coli)
- Bacteria produce insulin protein
- Insulin is purified for medical use 3. Biological Control and Bioremediation
Biological Control
Definition: Use of microorganisms to reduce plant disease or pests
Mechanisms:
● Compete with harmful organisms ● Produce toxins that kill pests
Example:
● Bacillus thuringiensis (Bt) produces toxins that kill insects
Bioremediation
Definition: Use of microorganisms to clean up environmental pollutants
Key Concepts:
● Biotransformation: chemical change of pollutant ● Biodegradation: breakdown into non-toxic products
Examples:
● Oil spill cleanup ● Degradation of pesticides and chemicals
4. Genetically Modified Animals
Transgenic Animals
Animals that carry genes from another species
Example:
● Salmon engineered to grow faster
Gene Modification vs Gene Addition
Gene modification (gene editing):
● Alters an existing gene
Gene addition:
● Blood clotting factors ● Antibodies ● Hormones
6. Reproductive Cloning
Definition
Producing genetically identical organisms
Dolly the Sheep (Important Example)
Steps:
- Remove nucleus from egg cell
- Insert nucleus from adult somatic cell
- Fuse cells using electricity
- Allow embryo to develop
- Implant into surrogate mother
Result:
● Clone genetically identical to donor
Issues with Cloning
● Possible premature aging (telomere shortening) ● Health problems ● Ethical concerns
7. Stem Cells
Key Characteristics
- Ability to divide (self-renew)
- Ability to differentiate into specialized cells
Types of Stem Cells
Totipotent
● Can form all cell types (entire organism)
Pluripotent
● Can form almost all cell types
Multipotent
● Can form several related cell types
Unipotent
● Can form only one cell type
Sources of Stem Cells
● Embryonic stem cells (ES cells) ● Embryonic germ cells (EG cells) ● Adult stem cells
Induced Pluripotent Stem Cells (iPS Cells)
● Adult cells reprogrammed into pluripotent state ● Avoid ethical issues of embryonic cells
Medical Uses of Stem Cells
● Repair damaged tissues ● Treat diseases such as: ○ Parkinson’s disease ○ Spinal cord injuries ○ Heart damage ○ Burns
Example:
● Bone marrow transplants
Ethical Concerns
● Source of embryonic stem cells ● Use of human embryos ● Cloning implications
Chapter 22:
1. Key Definitions
Cytogenetic → visual, low resolution Linkage → genetic crosses Physical → DNA-level precision
3. Cytogenetic Mapping and FISH
Cytogenetic Mapping
● Chromosomes stained to reveal banding patterns ● Used to locate genes on chromosomes
Fluorescence In Situ Hybridization (FISH)
Purpose:
● Locate a gene on a chromosome
Steps:
- Create DNA probe complementary to gene
- Label probe with fluorescent dye
- Hybridize probe to chromosome
- Visualize under fluorescence microscope
Chromosome Painting
● Multiple probes used ● Different chromosome regions appear in different colors
4. Linkage Mapping and Molecular Markers
Molecular Markers
● DNA sequences with known locations ● Used to track inheritance
Polymorphism
● Variation in DNA sequence between individuals
Types of Molecular Markers
Restriction Fragment Length Polymorphism (RFLP)
● Differences in DNA fragment lengths after restriction enzyme digestion
Microsatellites
● Short repeated sequences (e.g., CA repeats) ● Highly variable among individuals
Sequence Tagged Site (STS)
● DNA region amplified by PCR ● Used to identify microsatellites
Using Markers in Mapping
● Markers that are close together are inherited together ● Recombination frequency helps determine distance
Microsatellite Analysis
● PCR amplifies region around repeats ● Same repeat number → one band ● Different repeat numbers → two bands
Pedigree Analysis
● Track markers through families ● Helps identify location of disease genes
Comparison
SRS → accurate, cheap LRS → better assembly, handles repeats
7. Illumina Sequencing (Know This)
Type: Short-read sequencing
Also called: Sequence by synthesis
Basic idea:
● DNA is copied one base at a time ● Each base emits a fluorescent signal ● Machine records sequence
8. High-Throughput and Next-Generation Sequencing
High-throughput sequencing
● Rapid sequencing of large amounts of DNA
Next-generation sequencing
● Millions of sequences analyzed at once
9. Human Genome Project
Goals:
● Create linkage map ● Create physical map ● Sequence entire human genome ● Develop data analysis tools
Impact:
● Faster identification of disease genes ● Major advances in medicine
10. Metagenomics
Definition
Study of genetic material from environmental samples
Metagenome
● Collection of DNA from all organisms in sample
Strategy
- Collect environmental sample
- Extract DNA
- Perform shotgun sequencing
- Analyze sequences
Uses
Medicine
● Study microbiome
Agriculture
● Identify beneficial soil microbes
Bioremediation
● Find pollutant-degrading organisms
Biotechnology
● Discover new drugs
Environmental science
● Study ecosystems and global processes
Chapter 23:
1. Key Definitions
Functional Genomics
● Study of gene function across the entire genome
Proteome
● All proteins an organism can produce
3. RNA Sequencing (RNA-Seq)
What is RNA-Seq?
● A method to analyze gene expression using sequencing
Steps
- Isolate RNA
- Fragment RNA
- Convert to cDNA
- Sequence cDNA
- Align sequences to genome
What It Tells You
● Which genes are expressed ● How much each gene is expressed ● Alternative splicing patterns ● New RNA variants
Advantages over Microarrays
● More accurate ● Detects low-abundance RNA ● Identifies new transcripts ● Shows exon/intron boundaries
4. Gene Knockout Collections
What Are They?
● Collections of organisms where each has one gene inactivated
Purpose
● Determine function of genes ● Observe phenotype changes
How They Are Made
● CRISPR-Cas ● Transposable elements
Why Useful
● Study gene function on a large scale ● Identify gene pathways
5. Proteomics
Key Idea
● Proteome is larger than genome
Why?
One gene → multiple proteins due to:
- Alternative splicing
- RNA editing
- Posttranslational modifications
Types of Protein Modifications
Irreversible:
● Proteolytic cleavage ● Addition of groups (sugars, lipids)
Reversible:
● Phosphorylation ● Acetylation ● Methylation
