Understanding Positional Cloning: A Key Technique in Gene Mapping and Isolation

What is positional cloning is?
a. Using a selection procedure to clone a cDNA
b. Cloning a protein of a gene using PCR
c. Isolating a gene by PCR using primers from another species
d. Mapping a gene to a chromosomal region and then identifying and cloning the genomic copy of the gene from the region

 

Positional cloning is a powerful molecular biology technique used to identify and isolate genes based on their location on a chromosome. This method has been instrumental in discovering genes linked to genetic diseases and understanding complex biological traits. By mapping a gene to a specific chromosomal region, researchers can then identify and clone the genomic copy of the gene from that region.

---

Correct Answer: (d) Mapping a gene to a chromosomal region and then identifying and cloning the genomic copy of the gene from the region

The correct answer is (d) because positional cloning relies on the physical location of a gene within the genome. Unlike other cloning methods, which depend on the gene product or sequence, positional cloning focuses on gene mapping and identification.

---

What is Positional Cloning?

Positional cloning is a gene identification method that involves mapping a gene to a specific chromosomal region before isolating and cloning the genomic copy. This technique is essential for identifying disease-causing genes and understanding genetic inheritance patterns.

---

Keyphrase: Positional Cloning

Positional cloning involves several key steps, including linkage analysis, physical mapping, and gene isolation. This technique has significantly advanced human genetics and disease research.

---

Steps in Positional Cloning

1. Linkage Analysis

• Linkage analysis determines whether a gene is inherited along with specific genetic markers.
• Genetic markers, such as microsatellites and single nucleotide polymorphisms (SNPs), are used to identify chromosomal regions of interest.

---

2. Physical Mapping

• Once the gene’s approximate location is identified, physical mapping is conducted.
• Techniques such as fluorescence in situ hybridization (FISH) and radiation hybrid mapping are used.
• High-resolution physical maps help narrow down the gene’s location.

---

3. Gene Identification

• Candidate genes within the mapped region are identified.
• Molecular techniques such as Southern blotting, Northern blotting, and cDNA cloning are used.
• Candidate genes are tested for mutations or expression changes linked to the disease phenotype.

---

4. Cloning of the Gene

• The identified gene is cloned using PCR or bacterial artificial chromosomes (BACs).
• The cloned gene is sequenced to confirm its identity.

---

Significance of Positional Cloning

1. Disease Gene Discovery

• Positional cloning has helped identify genes responsible for genetic diseases like cystic fibrosis, Huntington’s disease, and Duchenne muscular dystrophy.
• By analyzing inheritance patterns and genetic markers, researchers can pinpoint disease-related mutations.

---

2. Identification of Genetic Markers

• Positional cloning allows the discovery of genetic markers linked to specific traits.
• These markers are used for genetic screening and diagnostic tests.

---

3. Understanding Gene Function

• Cloned genes can be studied to understand their role in biological processes.
• Functional analysis helps uncover the molecular basis of diseases and traits.

---

Techniques Used in Positional Cloning

1. Fluorescence In Situ Hybridization (FISH)

• FISH is used to visualize the location of a gene on a chromosome.
• Fluorescent probes bind to specific DNA sequences.

---

2. Genetic Linkage Mapping

• Linkage mapping identifies genetic markers inherited along with the target gene.
• Microsatellites and SNPs are used as markers.

---

3. Comparative Genomic Hybridization (CGH)

• CGH compares DNA samples to identify copy number variations.
• It is useful for identifying chromosomal deletions and duplications.

---

4. Sequence Tag Sites (STS) Mapping

• STS mapping uses unique DNA sequences as landmarks for physical mapping.
• This technique helps build high-resolution genomic maps.

---

Challenges in Positional Cloning

1. Complex Traits

• Polygenic diseases involve multiple interacting genes.
• Mapping these genes is difficult due to genetic complexity.

---

2. Genetic Heterogeneity

• The same phenotype can result from mutations in different genes.
• Distinguishing between causative and incidental mutations is challenging.

---

3. Large Genome Size

• The human genome is over 3 billion base pairs long.
• High-resolution mapping and sequencing are required for accurate gene identification.

---

Applications of Positional Cloning

1. Medical Genetics

• Identification of disease-causing mutations.
• Development of gene-based therapies.

---

2. Plant and Animal Breeding

• Identification of genes responsible for desirable traits.
• Improved crop yields and disease resistance.

---

3. Evolutionary Biology

• Comparison of homologous genes across species.
• Insight into evolutionary relationships and adaptation.

---

Comparison of Positional Cloning with Other Cloning Techniques

---

How to Improve Success in Positional Cloning

1  Use high-density genetic markers for better resolution.
2  Combine linkage analysis with physical mapping for accuracy.
3  Validate candidate genes using expression analysis and mutation screening.
4  Employ advanced sequencing techniques for faster gene identification.

---

Future of Positional Cloning

1. Next-Generation Sequencing (NGS)

• NGS enables whole genome sequencing with high accuracy.
• Positional cloning combined with NGS improves efficiency.

---

2. CRISPR-Based Functional Analysis

• CRISPR-Cas9 allows targeted gene editing.
• Positional cloning can be followed by functional validation using CRISPR.

---

3. Single-Cell Genomics

• Positional cloning at single-cell resolution.
• Useful for identifying rare genetic variants.

---

Conclusion

Positional cloning remains a vital tool in molecular biology and genetics. It has enabled the discovery of genes linked to genetic diseases, improved our understanding of gene function, and facilitated the development of gene-based therapies. With advancements in sequencing and mapping technologies, positional cloning is becoming more efficient and precise.

For expert guidance in molecular biology techniques like positional cloning, join Let’s Talk Academy — the leading institute for CSIR NET Life Science, IIT JAM, GATE Biotechnology, and DBT JRF preparation.

👉 Learn more about gene cloning at Let’s Talk Academy.

---

FAQs

Q1. What is the role of genetic markers in positional cloning?
Genetic markers help map the gene’s location by identifying co-segregation patterns.

Q2. How does FISH help in positional cloning?
FISH allows the visualization of gene location on chromosomes using fluorescent probes.

Q3. What are the main challenges in positional cloning?
Complex traits, genetic heterogeneity, and large genome size make positional cloning difficult.

---

This article was written with insights from Let’s Talk Academy, a top coaching institute for life sciences and biotechnology competitive exams.