Access Keys:
Skip to content (Access Key - 0)
Biology 230 - Molecules and Cells

Recombinant DNA Technology

Versions Compared

Key

  • This line was added.
  • This line was removed.
  • Formatting was changed.
Comment: Migration of unmigrated content due to installation of a new plugin

...

Section
Column
width35%
Panel

Wiki Markup
{align:center}
!image.jpg|thumbnail!
{align}
Align
aligncenter

Image Added

Figure 1. Restriction endonucleases cleave DNA at specific sites.Shown are the recognition sites for two restriction endonucleases: Eco RI and Alu I. The arrows indicate where the phosphodiester bonds will be broken. Both recognition sites are palindromes. Eco R1 has a 6 bp recognition site, and leaves sticky ends with 4-nucleotide overhangs. Alu I has a 4 bp recognition site, and leaves blunt ends.

Column
width65%

One of the first steps in being able to manipulate and study discrete DNA sequences is the ability to cleave large molecules of DNA into smaller defined fragments. This can be accomplished by the use of restriction endonucleases, which are enzymes that can cleave (or digest) double-stranded DNA in a site-specific fashion. These enzymes are found naturally in a variety of different bacteria, and are used by the bacteria to degrade foreign DNA; their own DNA is modified to be protected from degradation. Generally, restriction endonucleases recognize a short nucleotide sequence (4-8 bp) and cleave the DNA in a precise fashion. Cleavage breaks the phosphodiester bond in both of the two strands of double-stranded DNA. Many restriction endonuclease recognition sites are palindromic, meaning the sequence is identical whether reading from the top or bottom strand (but always reading 5' to 3'). For example, the restriction endonuclease Eco RI, which cleaves the sequence 5'GAATTC3', was initially isolated from bacteria (Escherichia coli). This restriction endonuclease will cleave DNA isolated from any cell, prokaryotic or eukaryotic. There are dozens of different restriction endonucleases that recognize and cleave different sequences. Examples of two restriction endonucleases and their recognition sites are illustrated in Figure 1. 

Some enzymes (e.g. Eco R1) cleave DNA in such a fashion as to leave short, single-stranded overhangs (sticky ends); others (e.g. Alu I) leave blunt ends(illustrated in Figure 1). If DNA, from any source (e.g. bacteria, plants or animals), is cleaved with a single restriction enzyme, the generated DNA fragments will have identical sequences at their ends. The frequency of cleavage of DNA is simply the frequency of a particular site occurring in the DNA. For an enzyme that has a 6 bp recognition site, the probability that the four nucleotides (G, A, T, C) will make up the recognition sequence is 1 in 4096 bp (1 in 46).

...

Section
Column
width50%
Panel

Wiki Markup
{align:center}
!image-1.jpg|thumbnail!
{align}
Align
aligncenter

Image Added

Figure 2. Agarose gel electrophoresis. An agarose gel is submerged in buffer in a gel box (forefront). The gel box is connected to a power supply (rear).

Column
width50%
Panel

Wiki Markup
{align:center}
!image-2.jpg|thumbnail!
{align}
Align
aligncenter

Image Added

Figure 3. A typical agarose gel. Above is an agarose gel stained with ethidium bromide and viewed under UV light. The left lane contains the molecular weight marker, with bands ranging from 1,000 bp (1 kb) to 10,000 bp (10 kb). The other lanes contain DNA that has been cleaved with a variety of enzymes (indicated at the top).

...

When the total nuclear DNA from a human cell is cleaved with Eco R1, it generates so many fragments of different sizes that when the fragments are separated by agarose gel electrophoresis they appear as a smear of DNA rather than individual bands. It would be impossible to determine if a particular band was present or perhaps altered. The technique of Southern blottingpermits the detection of a single fragment of DNA using a sequence-specific probe. DNA fragments are separated by gel electrophoresis and the bands are transferred to a special membrane that binds single-stranded nucleic acid. Typically the DNA is first denatured in the gel, by soaking it in alkaline buffer, and then it is rapidly neutralized. The gel is laid flat, the membrane is laid over the gel, and a stack of absorbent paper towels is placed on top of the membrane. The absorbent paper will wick the liquid from the gel through the membrane and, as it does so, the DNA will travel from the gel onto the membrane and be bound there. The relative positions of the DNA in the gel are preserved when the DNA is transferred to the membrane. The membrane, also referred to as the blot, is removed and then mixed with a specific probe. The probe is a single strand of DNA of a specific sequence, typically labeled fluorescently or radioactively. The membrane is placed in a solution with the probe and left for several hours. The probe will anneal (hybridize) to the fragments bound to the membrane that contain complementary DNA sequences. The unbound probe is washed away, and what remains on the blot is the labeled probe hybridized to the fragments that possess complementary DNA. Using this technique, one can detect the presence of a unique fragment of DNA in an entire fragmented genome.

Panel

Wiki Markup
{align:center}
!image-3.jpg|thumbnail!
{align}
Align
aligncenter

Image Added

Figure 4. RFLP in the beta globin gene in sickle cell anemia.The wild-type beta-globin gene (red) and the sickle cell anemia beta-globin gene (pink) are shown. The Dde I sites are marked by the stars. The mutation in the beta-globin gene results in the loss of one Dde I site. Genomic DNA is isolated from the cells of normal (wild type) sickle cell carriers (heterozygous for the sickle cell beta-globin gene - one wild-type gene and one sickle cell beta-globin gene) and sickle cell patients (homozygous for the sickle cell beta-globin gene - both genes are sickle cell beta-globin). Remember that cells are diploid and thus have two copies of each gene. The genomic DNA is cleaved with Dde I, the fragments are separated by gel electrophoresis and then transferred to the membrane. The membrane is hybridized to labeled beta-globin DNA. The wild-type gene has two fragments and the sickle cell gene has only one larger fragment.

Southern blotting and hybridization techniques have been employed to detect and screen for genetic diseases and disorders. For instance, the disease sickle cell anemia arises from a single nucleotide change in the beta-globin gene. This change in sequence affects the recognition site of the restriction endonuclease Dde I so that it can no longer cleave the DNA. This will change the Dde I cleavage pattern for the beta-globin gene. This is referred to as restriction fragment length polymorphism (RFLP). One can detect the particular RFLP associated with sickle cell anemia by preparing a Southern blot of DNA cut with Dde I and then hybridizing it to a probe specific for beta-globin (see Figure 4).

...

Section
Column
width35%
Panel

Wiki Markup
{align:center}
!image-4.jpg|thumbnail!
{align}
Align
aligncenter

Image Added

Figure 5.  A typical plasmid used for cloning.The origin of replication (ORI) is indicated in orange. The antibiotic resistance gene (Amp) is indicated in dark blue, and a region composed of multiple cloning sites is indicated in pale blue.

Column
width65%

Although the techniques described allow one to distinguish a region of DNA from the genome at large, it is even more useful to be able to isolate just the gene of interest and produce sufficient quantities of that particular region of the genome for additional investigations and manipulations. DNA cloning is the process of extracting just one region of the genome and producing many identical molecules of this DNA sequence. In order to do so, a recombinant DNA molecule (the DNA sequence of interest joined to a vector sequence) is constructed. The vectorsequence supports the replication and transmission of the recombinant DNA (usually in bacteria). Recombinant DNA molecules are created by mixing DNA fragments (e.g. fragments generated by Eco R1) from different sources (e.g. human and bacteria) that have compatible ends and then adding the enzyme ligase, which will join the two fragments of DNA. You may remember that DNA ligase joins the Okazaki fragments in DNA replication. In the test tube, isolated ligase will also join DNA fragments in a reaction termed ligation, as long as the ends are compatible (the same sticky ends or two blunt ends). Once the recombinant molecule is made it can be introduced into bacteria, where it will replicate and make many copies of this novel combination of DNA.

Most recombinant DNA molecules are made using bacterial plasmids as vectors. Plasmids are small (3,000-12,000 bp), circular DNA molecules that replicate in bacteria (Figure 5). All plasmids have an origin of replication (ORI), and many also encode for an antibiotic resistance (typically ampicillin resistance). Most plasmid vectors also have a number of different restriction endonucelase sites so that foreign DNA can be easily recombined with the plasmid. A recombinant DNA molecule can be introduced into bacteria through the process of transformation. The bacteria can be manipulated to take up DNA from the media in which they reside. Each bacterium takes up only one recombinant molecule. Typically the product of a ligation will be used to transform the bacteria. The frequency of transformation is low, but the few bacteria that have taken up recombinant DNA can be selected for easily by placing the bacteria in media with antibiotics (e.g. ampicillin) - only those with the recombinant DNA molecule (including the plasmid vector) will grow. The recombinant plasmid is present in hundreds of copies per bacterium, and is stably replicated and segregated to daughter cells each time the bacterium divides. Starting from a single bacterium, one can produce a large culture of bacteria that carries the identical recombinant plasmid. The bacterial culture can be lysed and the recombinant plasmid isolated from the rest of the bacterial DNA, providing a renewable source of pure recombinant plasmid.

The cloning of genes through this method has been supplemented by amplification of specific DNA sequences using the polymerase chain reaction (PCR, see the tutorial on DNA replication). Due to the availability of the complete genomic sequence of many organisms, it is relatively easy to design PCR primers specific for the amplification of any portion of the genome.

...