Once DNA cloning has provided us with large quantities of specific DNA segments, we can tackle some interesting questions about a particular gene and its function.
Any approaches for studying DNA molecules involve __, which uses a gel made of a polymer, such as a polysaccharide.
The gel acts as a molecular sieve to separate __ or __on the basis of __, __, and other physical properties.
Because nucleic acid molecules carry __charges on their __groups, They all travel toward the __pole in an electrical field.
- nucleic acids or proteins
- size, electrical charge
As they move, the thicket of polymer fibers impedes longer molecules more than it does shorter ones, separating them by length.
Thus, __separates a mixture of linear DNA molecules into bands, each consisting of DNA molecules of the same length.
One useful application of this technique is __, which can rapidly provide useful information about DNA sequences.
· In this type of analysis, the DNA fragments produced by __ (cutting) of a DNA molecule are sorted by __.
- restriction fragment analysis
- restriction enzyme digestion
- gel electrophoresis
When the mixture of restriction fragments undergoes __, it yields a band pattern characteristic of the starting molecule and the restriction enzyme used. In fact, the relatively small DNA molecules of viruses and plasmids can be identified simply by their restriction fragment patterns.
Because __can be recovered undamaged from gels, the procedure also provides a way to prepare pure samples of individual fragments- if the bands can be clearly resolved.
· Very large DNA molecules, like euk chromosomes, yield so many fragments that they appear as a smear instead of distinct bands.
__is also useful for comparing two different DNA molecules. A restriction enzyme recognizes a specific sequence of nucleotides, and a change in even one base pair of that sequence will prevent it from cutting at a particular site.
Restriction fragment analysis
Therefore, if the nucleotide differences between two __occur within a __, digestion with the enzyme that recognizes the site will produce a different mixture of fragments from each allele. Each mixture will give its own band pattern in __.
- restriction site
- gel electrophoresis
If we want to determine whether a person is a heterozygous carrier of the mutant allele for sickle-cell disease, we would directly compare the genomic DNA from that person with DNA from both a person with __ and a person who is __.
homozygous sickle cell disease homozygous for the normal allele.
__of genomic DNA digested with a restriction enzyme and stained with a DNA –biding dye yields too many bands to distinguish them individually.