DNA Sequencing & Gene Editing

Cheatsheet Content

### What is DNA Sequencing? DNA sequencing is the process of determining the exact order of nucleotides (A, T, G, C) in a DNA molecule. This order reveals genes, mutations, regulatory regions, and evolutionary relationships. #### Importance in Biology and Medicine - Transformed biology into an information-rich molecular science. - Aids in gene discovery, mutation detection, pathogen identification, evolutionary studies, cancer genetics, prenatal diagnosis, forensic science, and personalized medicine. - Connects genotype with phenotype, identifying disease-causing variants. #### Evolution of DNA Sequencing - Progression from chemical cleavage to enzymatic chain termination, then automated fluorescent systems, and modern high-throughput sequencing. - **Earliest practical methods (1977):** Maxam–Gilbert sequencing and Sanger’s dideoxy sequencing. - Sanger sequencing became dominant due to ease, safety, and automation adaptability. - Automated capillary sequencing powered the Human Genome Project. - Sequencing-by-synthesis (e.g., pyrosequencing) led to next-generation sequencing. #### Timeline Summary 1. Maxam–Gilbert (chemical cleavage) 2. Sanger dideoxy method (chain termination) 3. Automated fluorescent sequencing + capillary electrophoresis 4. Pyrosequencing and other sequencing-by-synthesis technologies 5. Modern genomics, genome mapping, precision medicine, gene editing ### Maxam–Gilbert Method A first-generation DNA sequencing technique based on chemical cleavage of DNA at specific bases. #### Principle - DNA fragment is 5′-end labeled with a radioactive marker. - Labeled DNA is divided into separate tubes and exposed to chemicals that cleave preferentially at specific bases (G, A+G, C, C+T). - Partial cleavage produces a series of labeled fragments of different lengths. - Fragments are separated by high-resolution polyacrylamide gel electrophoresis (PAGE). - Sequence is read from the band pattern (bottom to top, smallest fragments migrate farthest). #### In Short - Chemical sequencing - Base-specific cleavage - Radioactive labeling - Gel electrophoresis + autoradiography - Sequence inferred from fragment pattern #### Steps 1. DNA fragment selected. 2. 5′ end labeling with radioactive phosphate. 3. Denaturation to single-stranded DNA. 4. Division into 4 separate reaction tubes. 5. Base-specific chemical treatment (G), (A+G), (C), (C+T). 6. Partial cleavage of DNA. 7. Polyacrylamide gel electrophoresis. 8. Autoradiography. 9. Read sequence from bottom → top. #### Key Aspects - **Labeling:** Only one end is labeled, visualizing only fragments containing that end. - **Base-specific reactions:** Chemicals modify specific bases; piperidine breaks phosphodiester backbone at modified positions. - **Partial digestion:** Crucial to preserve sequence pattern. - **Electrophoresis:** Separates fragments differing by a single nucleotide. - **Reading:** Lane with band identifies base at that position. #### Maxam–Gilbert Sequencing Diagram ``` Labeled DNA strand │ ├── Tube 1 → chemical cleavage at G ├── Tube 2 → chemical cleavage at A+G ├── Tube 3 → chemical cleavage at C └── Tube 4 → chemical cleavage at C+T ↓ Fragment mixtures of different lengths ↓ Denaturing PAGE (4 lanes) -------------------------------- Lane G | Lane A+G | Lane C | Lane C+T -------------------------------- ↓ Autoradiograph band pattern ↓ Read sequence bottom → top ``` #### Advantages - Historically very important as one of the earliest methods. - Direct chemical interrogation of bases. #### Limitations - Uses hazardous chemicals. - Requires radioactive labeling. - Labor-intensive, difficult to automate. - Relatively short readable sequences. - Technically demanding compared to Sanger sequencing. **Summary:** Maxam–Gilbert sequencing is a chemical cleavage method where end-labeled DNA is partially broken at specific bases, and the sequence is read from electrophoretic band patterns. ### Sanger’s Dideoxy Method Also known as dideoxy chain termination, an enzymatic DNA sequencing technique where DNA synthesis is terminated by incorporation of ddNTPs (dideoxynucleotides). #### Principle - DNA polymerase extends a primer on a DNA template by adding normal deoxynucleotides (dNTPs). - If a ddNTP is incorporated, chain elongation stops because ddNTPs lack the 3′-OH group required for phosphodiester bond formation. - This creates a nested set of DNA fragments ending at every possible position of a given base. - After electrophoretic separation, the sequence is read from shortest to longest fragments. #### Why ddNTPs Terminate the Chain - **Normal dNTP:** has 3′-OH → next nucleotide can be added. - **ddNTP:** no 3′-OH → chain stops. #### Components - Template DNA - Primer - DNA polymerase - Four normal dNTPs: dATP, dGTP, dCTP, dTTP - Four chain-terminating ddNTPs: ddATP, ddGTP, ddCTP, ddTTP - Buffer + Mg²⁺ - **Classic method:** radioactive or labeled products. - **Modern method:** fluorescent dye-labeled ddNTPs. #### Steps ##### Classical Four-Tube Sanger Sequencing 1. Denature template DNA into single strands. 2. Anneal a primer complementary to known sequence. 3. Divide reaction into four tubes. 4. Each tube contains all four dNTPs plus one type of ddNTP. 5. DNA polymerase synthesizes new strands. 6. Incorporation of ddNTP stops elongation at specific positions. 7. Run products on polyacrylamide gel. 8. Read sequence from bottom upward. ##### Modern One-Tube Fluorescent Sanger Sequencing 1. Use all four fluorescent ddNTPs in one tube. 2. Generate fragments of varying lengths and colors. 3. Separate by capillary electrophoresis. 4. Detect fluorescence with a laser. 5. Software converts the signal into an electropherogram and base calls. #### Sanger Dideoxy Sequencing Flow ``` Template DNA + Primer ↓ DNA polymerase + dNTPs + small amount of ddNTPs ↓ Random chain termination at A / T / G / C positions ↓ Mixture of fragments of different lengths ↓ Electrophoresis ↓ Detection of terminal base ↓ Read DNA sequence ``` #### Sanger Dideoxy Sequencing Diagram ``` Template strand: 3' --------------------------- 5' Primer binds: ← Primer Extension by DNA polymerase: 5' ----A----T----G----C----A----T---- 3' | ddNTP inserted ↓ chain termination occurs Many fragments formed: 5' A 5' AT 5' ATG 5' ATGC 5' ATGCA 5' ATGCAT ↓ Size separation by gel/capillary ↓ Shortest fragment read first ↓ Sequence determined ``` #### Advantages - Highly reliable, accurate, relatively simple. - Easier to interpret than earlier methods. - Provides long read lengths for small DNA targets. - Still used for confirmatory sequencing, plasmid verification, and mutation checking. #### Limitations - Relatively slow, low-throughput, and expensive per base compared to modern methods. - Unsuitable for sequencing entire genomes economically. - Works best with relatively pure and simple DNA templates. #### Applications - Sequencing cloned DNA fragments - Mutation confirmation - Validation of variants found by NGS - Plasmid and construct verification - Small gene sequencing - Clinical and forensic confirmation in selected cases ### Automated DNA Sequencing Refers mainly to the fluorescent, instrument-based version of Sanger sequencing, where DNA fragments are separated by capillary electrophoresis and detected automatically by laser-induced fluorescence. #### Principle - Uses fluorescent dye-labeled terminators instead of manual gel lanes. - All fragments are generated in a single reaction. - Separated in capillaries filled with a replaceable polymer matrix. - Detected as they pass a laser detector. - Each terminal base emits a specific fluorescent signal, producing a colored chromatogram or electropherogram. #### Process 1. Cycle sequencing reaction with fluorescent ddNTPs. 2. Cleanup to remove excess dye terminators. 3. Capillary electrophoresis separates fragments by size. 4. A laser excites fluorescent dyes as fragments pass a detector. 5. A computer converts signals into colored peaks. 6. Sequence software performs base calling. #### Automated DNA Sequencing Flow ``` DNA sample ↓ Fluorescent Sanger reaction ↓ Mixture of dye-labeled terminated fragments ↓ Capillary electrophoresis ↓ Laser detection of fluorescent colors ↓ Computer generates chromatogram ↓ DNA sequence output ``` #### Key Concept: Capillary Electrophoresis - Replaced slab gels due to being faster, easier to automate, and compatible with multiple parallel capillaries. - Arrays of capillaries enabled high-throughput sequencing, crucial for projects like the Human Genome Project. #### Advantages - Higher throughput than manual Sanger. - Safer (no radioactive labeling). - Digital output and automated analysis. - Multiple samples can be run in parallel. - Long and accurate reads for targeted sequencing. #### Applications - Human Genome Project-era sequencing. - Clinical mutation analysis. - Microbial typing. - Forensic genetics. - QC of recombinant DNA constructs. - Small targeted sequencing projects. **Summary:** Automated DNA sequencing is fluorescent Sanger sequencing coupled with capillary electrophoresis and computerized detection. ### Pyrosequencing A sequencing-by-synthesis method where nucleotide incorporation is detected by the release of light, rather than by chain termination. #### Principle - When a nucleotide is incorporated, pyrophosphate (PPi) is released. - PPi is enzymatically converted into ATP. - ATP drives luciferase to produce light. - The amount of light is proportional to the number of nucleotides incorporated. - Sequence is determined by monitoring light signals as nucleotides are flowed over the template one at a time. #### Components (Enzymes and Substrates) ##### Enzymes - DNA polymerase - ATP sulfurylase - Luciferase - Apyrase ##### Other Components - Single-stranded DNA template - Sequencing primer - dNTPs added sequentially - APS (adenosine 5′ phosphosulfate) - Luciferin #### Role of Each Enzyme - **DNA polymerase:** adds the correct nucleotide. - **ATP sulfurylase:** converts PPi + APS into ATP. - **Luciferase:** uses ATP to convert luciferin into oxyluciferin, releasing light. - **Apyrase:** degrades leftover nucleotides and ATP before the next cycle. #### Steps 1. Primer anneals to a single-stranded DNA template. 2. One type of nucleotide is added. 3. If complementary, DNA polymerase incorporates it. 4. Incorporation releases PPi. 5. ATP sulfurylase converts PPi to ATP. 6. Luciferase uses ATP to generate light. 7. Light is measured as a peak in a pyrogram. 8. Apyrase removes excess nucleotides and ATP. 9. Next nucleotide is added. #### Pyrosequencing Flow ``` Template + Primer ↓ Add one nucleotide type ↓ If complementary → nucleotide incorporated ↓ PPi released ↓ PPi + APS --(ATP sulfurylase)→ ATP ↓ Luciferin --(luciferase + ATP)→ Light ↓ Light detected as peak ↓ Apyrase removes unused reagents ↓ Next nucleotide added ``` #### Pyrosequencing Reaction Diagram ``` DNA template + primer ↓ dNTP added one by one ↓ Correct nucleotide incorporated by DNA polymerase ↓ Pyrophosphate (PPi) released ↓ PPi + APS --ATP sulfurylase→ ATP ↓ Luciferin --luciferase + ATP→ Oxyluciferin + LIGHT ↓ Camera/sensor records light peak ↓ Apyrase degrades extra dNTPs and ATP ↓ Repeat cycle ``` #### Advantages - Rapid, real-time, gel-free. - Useful for short read analysis, SNP detection, and methylation studies. - Can process many reads in parallel, key for high-throughput sequencing. #### Limitations - Difficulty in accurately measuring homopolymer regions (e.g., AAAA), as light intensity may not scale perfectly. - Read lengths generally shorter than traditional Sanger reads. - Data interpretation can be harder in repetitive regions. #### Applications - SNP genotyping - Mutation detection - DNA methylation analysis - Microbial community analysis - Pathogen identification - Short targeted sequencing **Summary:** Pyrosequencing is a sequencing-by-synthesis method that detects nucleotide incorporation through enzymatic conversion of PPi into a measurable light signal. ### Human Genome Mapping The process of determining the relative positions of genes, markers, and important sequences on chromosomes. Includes genetic linkage maps, physical maps, and sequence maps (exact nucleotide order). Crucial step toward full genome sequencing. #### Human Genome Project (HGP) - **Duration:** 1990–2003. - **Goal:** International collaborative effort to map and sequence the human genome. - **Outcome:** Produced the first draft genome in 2000 and an essentially complete reference sequence in 2003. - Also sequenced model organisms, promoted open data sharing, and addressed ethical, legal, and social implications (ELSI). #### Why Mapping Came Before Full Sequencing - Scientists needed to know where important markers and clones were located before sequencing an entire genome. - Mapping helped organize chromosomes into ordered regions for systematic sequencing. Mapping provided the "road map"; sequencing provided the "exact text." #### Findings - **Genome size:** ~3 billion base pairs across 23 pairs of chromosomes. - **Gene number:** Surprisingly, humans have fewer genes than expected (~20,000 to 25,000). Biological complexity is explained by gene regulation, alternative splicing, non-coding DNA, and interaction networks, not just gene number. #### Additional Significance - Proved that large-scale, data-driven "big science" could succeed in biology. - Accelerated sequencing technology development, data analysis methods, and genome databases. #### Implications in Health and Disease 1. **Disease Diagnosis:** Improved by linking diseases to specific genes, variants, and molecular pathways (inherited disorders, cancer, prenatal diagnosis, rare diseases). Tumors can be profiled molecularly. 2. **Personalized Medicine:** Genomic information supports tailored prevention, diagnosis, and treatment. Genetic testing identifies disease susceptibility and guides drug selection/dosage (pharmacogenomics). 3. **Drug Development:** Expanded known molecular targets for therapy. Genomic data identifies disease-related genes, proteins, pathways, and biomarkers, leading to "designer drugs." 4. **Gene Therapy:** Knowledge of the genome improved gene therapy by identifying defective genes and understanding therapeutic correction needs. 5. **Ethical, Legal, and Social Implications:** HGP recognized concerns about privacy, discrimination, informed consent, and equitable access, establishing the ELSI program. ### RNA Interference (RNAi) A conserved biological process where small RNA molecules suppress gene expression by targeting messenger RNA (mRNA) for degradation or translational repression. A major tool for studying gene function and a natural regulatory mechanism. #### Principle - Sequence-specific gene silencing. - A small RNA with sequence complementarity to a target mRNA guides a protein complex (RISC) to that mRNA, reducing protein production. - Experimentally triggered using siRNA or shRNA. #### Mechanism (Steps) 1. Entry of dsRNA / shRNA precursor into the cell. 2. **Dicer** (RNase III-like enzyme) cleaves long dsRNA into siRNAs (~21–25 nucleotides). 3. siRNAs are loaded into the **RISC** (RNA-induced silencing complex). 4. The siRNA duplex is unwound; the guide strand is retained, and the passenger strand is removed. 5. The guide strand directs RISC to a complementary target mRNA. 6. If complementarity is high, **Argonaute/AGO2** within RISC cleaves the mRNA. 7. The cleaved mRNA is degraded, leading to reduced protein synthesis. #### siRNA vs miRNA - **siRNA:** Usually pairs almost perfectly with target mRNA, often causing direct cleavage. - **miRNA:** Usually pairs imperfectly, often causing translational repression or destabilization rather than exact cleavage. (Both are part of the broader RNA silencing framework.) #### Mechanism Diagram ``` Long dsRNA / shRNA ↓ Dicer ↓ siRNA duplex ↓ Loading into RISC ↓ Passenger strand removed ↓ Guide strand retained ↓ Guide strand pairs with target mRNA ↓ AGO2-mediated cleavage / silencing ↓ mRNA degraded ↓ Protein expression decreases ``` #### Applications - Functional genomics and gene knockdown studies. - Studying essential genes through partial silencing. - Drug target identification. - Antiviral and anticancer research. - Potential therapeutics based on RNA silencing. #### Advantages - Highly useful for loss-of-function studies. - Does not require permanent genome alteration. - Can produce partial knockdown, useful when complete knockout is lethal. #### Limitations - Knockdown may be incomplete. - Off-target silencing may occur. - Effects are often transient unless stable expression systems are used. - Silences gene expression but does not edit DNA sequence. ### Gene Editing Technologies #### Zinc Finger Nucleases (ZFN) Engineered nucleases made by fusing a zinc finger DNA-binding domain to a FokI nuclease cleavage domain. They create site-specific DNA double-strand breaks (DSBs). ##### Structure - **DNA-binding region:** Composed of zinc finger motifs; each finger recognizes ~3 base pairs. - **DNA-cleavage region:** FokI endonuclease domain, which cuts DNA only when two FokI domains dimerize. ##### Mechanism - Two ZFN monomers bind opposite DNA strands at neighboring target sequences separated by a spacer. - FokI nuclease domains dimerize and create a DSB. - Cell repairs the break by: - **NHEJ (non-homologous end joining):** Often causes insertions/deletions, producing gene disruption. - **HDR (homology-directed repair):** Can introduce precise edits if a donor template is supplied. ##### ZFN Mechanism Diagram ``` ZFN-L binds ← target DNA spacer → ZFN-R binds \ / \__ FokI dimerizes _____/ ↓ Double-strand break ↓ NHEJ or HDR repair ``` ##### Advantages - Targeted genome editing. - Can mediate both knockout and precise correction. - Earlier therapeutic genome-editing platform. ##### Limitations - Difficult to design (context-dependent zinc-finger interactions). - May show off-target cleavage and can be cytotoxic. - Engineering is laborious. ##### Applications - Gene knockout. - Correction of disease-causing mutations. - CCR5 disruption for HIV research. - Experimental gene therapy and model creation. #### TALENs Transcription Activator-Like Effector Nucleases (TALENs) are engineered proteins where a TALE DNA-binding domain is fused to the FokI nuclease domain. ##### Structure - TALE proteins from *Xanthomonas* bacteria. - DNA-binding domain: repeated modules (~33–35 amino acids), each recognizing one base pair via specific repeat-variable diresidues (RVDs). This "one-repeat/one-base" logic simplifies design. ##### Mechanism - Similar to ZFNs, TALENs work in pairs. - Left and right TALEN bind opposite sides of the DNA target. - FokI domains dimerize in the spacer region and create a DSB. - Repair proceeds through NHEJ or HDR. ##### TALEN Mechanism Diagram ``` TALEN-L binds target spacer TALEN-R binds target \ / \____ FokI dimerization _________/ ↓ Double-strand break ↓ NHEJ / HDR repair ``` ##### Advantages - Easier targeting logic than ZFNs. - High specificity. - Broad target range. - Useful in plants, animals, and diverse cell types. ##### Limitations - Large and repetitive proteins, making cloning and delivery difficult. - Assembly can be labor-intensive. - Vector packaging can be challenging. ##### Applications - Targeted mutagenesis. - Gene insertion and correction. - Crop improvement. - Model organism engineering. - Functional genomics. #### CRISPR-Cas System A genome-editing system from bacterial/archaeal adaptive immunity. CRISPR-Cas9 is the most common form: an RNA-guided nuclease system that introduces sequence-specific DNA breaks. ##### Components - **Cas9 nuclease.** - **Guide RNA:** - Naturally: crRNA + tracrRNA. - Engineered form: single guide RNA (sgRNA). - Target DNA. - **PAM (Protospacer Adjacent Motif):** Usually NGG for *S. pyogenes* Cas9. - Optional donor DNA template for HDR-based precise editing. ##### Principle - Unlike ZFN and TALEN (which require engineering new proteins for each target), CRISPR usually only requires changing the guide RNA sequence. - This makes CRISPR simpler, faster, and cheaper to retarget. ##### Mechanism (Stepwise) 1. Design sgRNA complementary to the target DNA sequence. 2. Cas9 binds sgRNA to form an active ribonucleoprotein complex. 3. The complex scans DNA for a valid PAM. 4. Once PAM is recognized, DNA near the PAM unwinds. 5. The sgRNA base-pairs with the complementary target DNA. 6. Cas9 cleaves both DNA strands, creating a DSB. 7. The cell repairs the break by: - **NHEJ:** causes indels → gene knockout. - **HDR** with donor template: precise substitution/insertion/correction. ##### Important Molecular Detail - Cas9 contains two nuclease activities (HNH and RuvC-like domains) that cut opposite DNA strands. - The break is usually produced a few nucleotides upstream of the PAM. ##### CRISPR-Cas9 Genome Editing Diagram ``` sgRNA = 20-nt guide sequence + scaffold + Cas9 ↓ Cas9–sgRNA complex formed ↓ Searches genome for PAM (NGG) ↓ PAM found next to target DNA ↓ Guide RNA base-pairs with target ↓ Cas9 cuts both DNA strands ↓ Double-strand break (DSB) / \ / \ NHEJ HDR ↓ ↓ Indels / knockout Precise correction ``` ##### More Detailed Mechanism Diagram ``` Target DNA: 5' ---- target sequence ---- NGG ---- 3' ↑ PAM Step 1: Cas9-sgRNA recognizes PAM Step 2: DNA unwinds Step 3: sgRNA binds complementary target Step 4: Cas9 cuts both strands Step 5: Cell repairs by NHEJ or HDR ``` ##### Advantages - Simple to design, highly versatile, relatively inexpensive, efficient, and multiplexable. - Multiple genes can be targeted simultaneously. - Adaptable for CRISPR interference, activation, base editing, etc. ##### Limitations - Off-target effects can occur. - PAM requirement restricts target choice. - Editing efficiency varies by locus and cell type. - Delivery of Cas proteins and guide RNAs can be challenging. - Mosaicism may arise in embryos/animals. - Serious ethical concerns, especially in human germline editing. ##### Applications - Gene knockout and knock-in. - Functional genomics. - Disease models. - Experimental gene therapy. - Agricultural trait improvement. - Synthetic biology. - Precision medicine research. ### Comparison Table | Method | Type | Key Feature | Main Principle | Major Advantage | Major Limitation | Typical Use | |---|---|---|---|---|---|---| | Maxam–Gilbert | Chemical sequencing | Base-specific chemical cleavage | End-labeled DNA is chemically cleaved at specific bases | Historically important; direct chemistry | Hazardous, radioactive, hard to automate | Early DNA sequencing | | Sanger | Enzymatic sequencing | ddNTP chain termination | DNA synthesis stops when ddNTP is incorporated | Very reliable and accurate | Slow and low-throughput for large genomes | Small-scale sequencing, validation | | Automated DNA sequencing | Automated fluorescent Sanger | Capillary electrophoresis + fluorescent detection | Dye terminators separated in capillaries and detected by laser | Faster, safer, digital output | Still limited throughput vs modern NGS | Clinical and project sequencing | | Pyrosequencing | Sequencing-by-synthesis | Light signal from PPi release | Nucleotide incorporation generates measurable light | Rapid and real-time | Homopolymer errors, short reads | SNPs, methylation, short reads | | RNAi | Gene silencing | Small RNA-guided mRNA suppression | siRNA/miRNA guides RISC to target mRNA | No DNA cutting required | Usually temporary/incomplete silencing | Functional genomics | | ZFN | Genome editing | Zinc-finger protein + FokI | Protein-guided DSB followed by NHEJ/HDR | Targeted editing possible | Complex protein engineering | Early targeted gene editing | | TALEN | Genome editing | TALE repeats + FokI | DNA-binding repeats guide DSB formation | High specificity, easier code than ZFN | Large constructs, delivery issues | Plants, animals, gene targeting | | CRISPR-Cas9 | Genome editing | RNA-guided targeting | sgRNA guides Cas9 to cut DNA near PAM | Simple, cheap, efficient, multiplex | Off-target risk, ethics, PAM dependence | Modern genome engineering | ### Diagram Practice Page #### 1. Sanger Method Flow ``` Template + primer ↓ DNA polymerase + dNTPs + ddNTPs ↓ Random chain termination ↓ Fragments of different lengths ↓ Electrophoresis / capillary run ↓ Read sequence ``` #### 2. Pyrosequencing Flow ``` Template + primer ↓ Add dNTP ↓ If incorporated → PPi released ↓ PPi → ATP (ATP sulfurylase) ↓ ATP + luciferin → light (luciferase) ↓ Apyrase resets system ``` #### 3. RNAi Mechanism Flow ``` dsRNA / shRNA ↓ Dicer ↓ siRNA ↓ RISC loading ↓ Guide strand binds mRNA ↓ mRNA cleavage / silencing ``` #### 4. CRISPR-Cas9 Flow ``` sgRNA + Cas9 ↓ PAM recognition ↓ Target DNA binding ↓ Double-strand break ↓ NHEJ / HDR ``` #### Labeling Tips for Scoring Better In diagrams, always label: - enzyme name - DNA/RNA template - product formed - where cleavage occurs - final output ### Quick Revision Box - **Maxam–Gilbert:** chemical cleavage - **Sanger:** ddNTP chain termination - **Automated sequencing:** fluorescent Sanger + capillary electrophoresis - **Pyrosequencing:** light from PPi release - **RNAi:** post-transcriptional gene silencing - **ZFN/TALEN:** protein-guided nucleases - **CRISPR-Cas9:** RNA-guided nuclease - **Genome mapping:** locating genes/markers on chromosomes