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 __on the basis of __, __, and other physical properties.
nucleic acids or proteins
size, electrical charge
Because nucleic acid molecules carry __charges on their __groups, They all travel toward the __pole in an electrical field.
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
When the mixture of restriction fragments undergoes __, it yields a band pattern characteristic of the starting molecule and the __used. In fact, the relatively small DNA molecules of viruses and plasmids can be identified simply by their __.
restriction fragment patterns
True or False:
Because DNA 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 __.
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 __yields too many bands to distinguish them individually.
However, a method called __, which combines gel electrophoresis and nucleic acid hybridization, allows us to detect just those bands that include parts of the β-globin gene.
DNA –binding dye
The principle is the same as in__ for screening bacterial clones.
In __, the probe is usually a radioactive single-stranded DNA molecule that is complementary to the gene of interest.
The identification of carriers of mutant alleles associated with genetic diseases is only one of the ways __ has been used.
nucleic acid hybridization
Southern blotting x2
In fact, this technique has been a lab workhouse for many years Recently, however, it has been supplanted by more rapid methods, often involving __ of the specific parts of genomes that may differ.
Once a gene is cloned, its complete nucleotide sequence can be determined.
· The automated procedure for this is based on a technique called the __.
o This method was developed by British biochemist Frederick Sanger.
dideoxyribonucleotide (or dideoxy, for short) chain termination method
True or False:
Knowing the sequence of a gene allows researchers to compare it directly with gene in other species, where the function of the gene product may be known.
If two genes from different species are quite similar in sequence, it is reasonable to suppose that their gene products perform similar functions. In this way, sequence comparisons provide clues to a gene’s function.
Having cloned a given gene, researchers can make labeled __ that will hybridize with mRNAs transcribed from the gene. The probes can provide information about when or where in the organism the gene is transcribed. Transcription levels are commonly used as a measure of __.
nucleic acid probes
If we wanted to find out how the expression of the β-globin gene changes during the embryonic development of the hummingbird, there are at least two ways:
__ and __
Reverse transcriptase- polymerase chain reaction, or RT-PCR
__: carry out gel electrophoresis on samples of mRNA from hummingbird embryos at different stages of development, transfer the samples to a nitrocellulose membrane, and then allow the mRNAs on the membrane to hybridize with a labeled probe recognizing β-globin mRNA. If we expose a film to the membrane, the resulting image will look similar to the Southern blot, with one band of a given size showing up in each sample. If the mRNA band is seen at a particular stage, we can hypothesize that the protein functions during events taking place at that stage. Like Southern blotting, it has been a mainstay over the years, but it is being supplanted in many labs by other techniques.
__: quicker and more sensitive
o Analysis of hummingbird β-globin gene expression with this begins similarly to Northern blotting, with the isolation of mRNAs from different developmental stages of hummingbird embryos. Reverse transcriptase is added next to make cDNA, which then severs as a template for PCR amplification using primers from the β-globin gene. When the products are run on a gel, copies of the amplified region will be observed as bands only in samples that originally contained the β-globin mRNA.
o In the cause of hummingbird β- globin, we might expect to see a band appear at the stage when RBCs begin forming, with all subsequent stages showing the same band. RT-PCR can also be carried out with mRNAs collected from different tissues at one time carried out with mRNAs collected from different tissues at one time to discover which tissue is producing a specific mRNA.
Reverse transcriptase- polymerase chain reaction, or RT-PCR
An alternative way to determine which tissues or cells are expressing certain genes is to track down the location of specific mRNAs using labeled probes in place, or __, in the intact organism.
· This technique, called __, is most often carried out with probes labeled by attachment of fluorescent dyes. Different probes can be labeled with different dyes.
in situ hybridization
True or False:
Researchers use genome sequences as probes to investigate which genes are transcribed in different situations, such as in different tissues or at different stages of development. They also look for groups of genes that are expressed in a coordinated manner, with the aim of identifying networks of gene expression across an entire genome.
The basic strategy in such global (genome-wide) expression studies is to isolate the mRNAs made in particular cells, use these molecules as templates for making the __by __, and then employ __to compare this set of __with a collection of DNA fragments representing all or part of the genome.
True or False:
The results identify the subset of genes in the genome that are being expressed at a given time or under certain conditions. DNA technology makes such studies possible; with automation, they are easily performed on a large scale.
Genome-wide expression studies are made possible by __.
· A __consists of tiny amounts of a large number of single-stranded DNA fragments representing different genes fixed to a glass slide in a tightly spaced array, or grid.
· __ are also called __.
· Ideally, these fragments represent all the genes of an organism.
DNA microarray assays
In __on nematodes, results show that expression of nearly __% of the genes changes dramatically during development and that many genes are expressed in a sex-specific pattern.
· This supports the model that embryonic development involves a complex and elaborate program of __, rather than simply the expression of a small number of important genes.
In addition to uncovering gene interactions and providing clues to gene function, __may contribute to a better understanding of diseases and suggest new diagnostic techniques or therapies.
Information from __ should provide a grander view of how ensembles of genes interact to form an organism and maintain its vital systems.
DNA microarray assays x2
Determining function of a gene:
· Most common approach: __, specific mutations are introduced into a cloned gene, and then the mutated gene is returned to a cell in such a way that it disables (“__”) the normal cellular copies of the same gene. If the introduced mutations alter or destroy the function of the gene product, the phenotype of the mutant cell may help reveal the function of the missing normal protein.
· Using molecular and genetic techniques worked out in the 1980s, researchers can even generate mice with any given gene disabled, in order to study the role of that gene in development and in the adult.
in vitro mutagenesis
A newer method for silencing expression of selected genes exploits the phenomenon of __.
· This experimental approach uses synthetic double-stranded RNA molecules matching the sequence of a particular gene to trigger breakdown of the gene’s mRNA or to block its translation.
· Has been used successfully to reduce (“__”) the expression of specific genes in mammalian cells, including human cells, and plans are under way to try for the treatment of certain human disorders, including macular degeneration of the eye.
· In other organisms, __has already proved valuable for analyzing the functions of genes on a large scale.
· In one study, it was used to prevent expression of __% of the genes in early nematode embryos, one gene at a time.
RNA interference (RNAi)
DNA research is being augmented by the development of powerful techniques for cloning whole multicellular organisms.
· Aim of this work is to obtain special types of cells, called __, that give rise to all the different kinds of tissues.
o On a basic level, __would allow scientists to use the DNA methods to study the process of cell __.
o On a more applied level, DNA techniques could be used to alter stem cells for the treatment of disease.