It helps in isolating the non-contiguous clones from a genomic library. These clones skip a region between the known points on a chromosome. Chromosome jumping acts as a tool for bypassing the regions that do not allow the cloning process. It plays a crucial role in physical mapping techniques for generating genomic markers. Chromosome jumping helps in searching for a specific gene. The regions difficult to clone include certain repetitive sequences. It helps in analyzing the sequences separated by more than 100-kilobases. Chromosome jumping involves two alternative procedures. The first method uses rare cutting enzymes. The second method uses frequent cutters. Chromosome jumping is also known as chromosome hopping. Consider the phage DNA.
A method using the rare cutters:
These cutters belong to the class of endonucleases. The first step involves the isolation of the genomic DNA. The isolated DNA gets a treatment with the restriction enzyme (rare cutter). The process is known as restriction digestion. It allows the isolation of fragments with 100 kb sizes. These fragments get subjected to a specialized electrophoretic technique known as the pulse field gel electrophoresis. The separated fragments get easily circularized later on. This method gives rise to three types of fragments. The first type includes the fragments having sequences with 100-kilobase sizes. The second type of fragments includes sequences of the original augments with lighted sequences such as the junction fragments. The third type of fragments includes other types of genomic sequences.
A method using frequent cutters:
The frequently cutting endonucleases lead to partial digestion of the fragments. First, the genomic DNA gets isolated from the sample. Next, the restriction enzymes carry out partial digestion of the genomic DNA. The fragments obtained from the partial digestion get selected based on the size. The next step involves the pulse field gel electrophoresis. The fragments obtained from the pulse field gel electrophoresis get cloned into a suitable vector.
Cloning:
The fragments obtained from both the procedures get cloned into a suitable vector with the presence of a selectable marker. The vector gets introduced into the host such as E. coli bacteria. After this, the cells get plated on a medium to check the growth of the plaques. The bacteriophages genome having the selectable marker gets replicated to form the plaques. They are known as the jumping clones. Next, the jumping fragments get identified through the nucleic acid hybridization. It involves using a specific probe. Thus, it is possible to identify the fragments undergoing repetitive hopping process.
Image: Chromosome jumping library depicting jumping clones
Chromosome jumping library:
It includes a collection of recombinant molecules obtained through chromosome jumping. The two types of jumping libraries include the general jumping and the specific jumping libraries. The general jumping libraries include sequences starting at any genomic locations. They travel to specific distances. These libraries involve sequences obtained from chromosome jumping involving frequent cutters. The specific jumping libraries show a specific jumping of the clones. They jump from the rare restriction site to the adjacent restriction sites. These libraries involve sequences obtained from the chromosome jumping methods using the rare cutters. The examples include libraries constructed using Not-I restriction endonucleases. Thus, with the help of chromosome jumping libraries, the clones jump from one restriction site to the other.
Chromosome jumping libraries have many applications. They include prenatal diagnosis, de novo assembly, and characterizing chromosomal rearrangements. Highly efficient bacterial genome assemblies were constructed using long jump chromosome libraries. The whole genome jumping library offers a gene-level resolution. Thus, it is beneficial for prenatal diagnosis and testing. A short jump library gets created using ligation of the DNA fragments with biotinylated, followed by circularization and affinity assays. However, it is less efficient due to its reduced genome coverage. Long jump library is efficient for longer DNA fragments. Another type of chromosome jumping library known as custom barcode jumping library distinguishes the junction fragments very efficiently. The E. coli vector transfection helps in amplifying larger DNA fragments in the Fosmid-jump library.
Enzymes used in chromosome jumping:
An endonuclease enzyme shows capability in cleaving the phosphodiester bond in the DNA strand. The technique of chromosome jumping involves restriction enzyme such as endonuclease type-II. These enzymes efficiently cleave longer DNA sequences. The following table depicts the examples of endonucleases used in chromosome jumping:
Endonuclease
|
Restriction site
|
Not I
|
GCGGCCGC
|
Sfi I
|
GGCCNNNNNGGCC
|
Pac I
|
TTAATTAA
|
BssHII
|
GCGCGC
|
Table: Endonucleases used in chromosome jumping and their restriction sites
Certain regions in the mammalian genome show the presence of rare nucleotide repeats. Such regions also get considered through enzyme treatment.
Genetic disorders arise due to defective genes or mutations. However, certain genetic disorders, the mutant genes or the gene products remain unidentified. In such cases, it becomes difficult to know the exact details of the genes and the pathways involved. Thus, identifying these genes and cloning them becomes the most difficult task. Also, the molecular markers prove to be inefficient in identifying such genes. The reason involves very large molecular distances. Reverse genetics is a subfield of genetics involving investigation of a gene or a protein function. The first step involves directed mutagenesis using the knowledge of a DNA or a protein sequence. Next, the programmed mutations get introduced back to the genome. However, it is difficult to identify the unknown sequences responsible for causing a genetic disorder. The problem could be solved using a chromosome jumping library. A review mentioned the chromosome jumping library constructed for the cloning of DNA sequences. These sequences lie a hundred kilobases away from the gDNA start point.
References:
[1] Encyclopedia of Genetics, Genomics, Proteomics, and Informatics, by George P. Rédei
[2] The Dictionary of Genomics, Transcriptomics, and Proteomics, By Guenter Kahl
[3] Plant Chromosomes, by Archana Sharma
[4] Introduction to Plant Biotechnology, by H. S. Chawla
[5] Molecular Biology and Genetic Engineering, by P. K. Gupta
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