Which of the following characteristic of Figure 1 best shows that the fragment is RNA and not DNA?

Isolation and Characterization of DNA Fragments Using Gel Electrophoresis

Once DNA fragments have been generated using restriction enzymes or PCR amplification, they must be purified and separated from other DNA fragments that are not of interest. The separation and identification of DNA fragments based on their size is possible using a ubiquitous tool called gel electrophoresis. Gel electrophoresis is used to isolate, identify, and characterize properties of DNA fragments (Figure 10.4).

First, a scientist uses molds to make an agarose gel with wells at one end for placing DNA samples. The gel is placed in an electrophoresis chamber and DNA samples are added to the wells. The electrophoresis apparatus produces a small electrical field, driving negatively charged DNA strands away from the cathode (the negative end) and toward the anode (the positive end). The mobility of the DNA fragments through the gel is inversely proportional to the logarithm of the number of base pairs in the fragments. Therefore, smaller DNA fragments will migrate much more quickly through the gel than large fragments. One of the lanes of the gel typically contains a DNA ladder—DNA that has been previously digested and characterized, producing fragments with known base pair sizes. Therefore, it is possible to determine the sizes of the fragments in the DNA samples by comparing their location to the location of the fragments in the ladder.

The DNA bands on agarose gels are invisible unless the DNA is labeled or stained. A common method of staining agarose gels is briefly incubating them in a solution containing ethidium bromide, a DNA intercalating agent that fluoresces under ultraviolet light when bound to DNA. After identifying DNA fragments in a gel, it is possible to physically cut the DNA fragments out of the gel and purify the DNA from the agarose using simple, commercially available kits and protocols. After purifying the DNA, a scientist can clone the fragment into a storage vector.

2.1 DNA Constructs Used for Four-Protein Coexpression in E. coli

DNA fragments encoding residues 35–168 of ISCU, which comprise the predicted mature form of this protein (Tong & Rouault, 2000), and residues 42–210 of FXN, which comprise the longest native isoform of FXN (Gakh et al., 2010), are PCR amplified and cloned into the pET-52b(+) 3C/LIC vector using the LIC Duet Adapter and LIC Cloning kit (Novagen) using a protocol provided by the manufacturer. This cloning enables the simultaneous expression of ISCU and FXN42–210, the latter protein fused to an N-terminal Strep•Tag® II (Fig. 6A). It should be noted that the positioning of the Strep•Tag® II-FXN42–210 DNA fragment upstream of the ISCU DNA fragment results in robust expression of both proteins, whereas the opposite orientation yields very low levels of FXN42–210. DNA fragments corresponding to residues 56–458 of NFS1 and 6–92 of ISD11, comprising the predicted mature forms of these proteins (Li et al., 2009; Marelja, Stocklein, Nimtz, & Leimkuhler, 2008), are, respectively, cloned into the BglII and HindIII sites and the NcoI and EcoRI sites of the pCDFDuet-1 vector (Novagen). This cloning enables the simultaneous expression of ISD11 and NFS1, the latter protein fused to an N-terminal six-histidine tag (Fig. 6A). The two plasmids encoding FXN42–210 and ISCU, and NFS1 and ISD11, are cotransformed in the E. coli strain BL21 (DE3) (Novagen) for four-protein coexpression.

2.2 Cloning procedure

A DNA fragment encoding CBM3a (CipA residues 368–522) was amplified by PCR from C. thermocellum ATCC27405 genomic DNA using the following primers:

F-CATATGGCAAATACACCGGTATC

R-GGATCCTATTTACCCCATACAAGAACACC

PCR products were purified and cleaved with the restriction enzymes NdeI and BamHI and inserted into the pET-28a(+) expression vector (Novagen, Madison, WI) together with an N-terminal hexahistidine tag.

DNA fragments encoding cohesin 3 with (Linker-Coh3) and without (Coh3) the adjacent linker (CipA residues 543–702 and residues 561–702, respectively) were amplified by PCR from C. thermocellum ATCC27405 genomic DNA, using the following primers:

F-GAATTCGAACCCGGTGGCAGTGTAG (Linker-Coh3)

F-GAATTCAATGCAATAAAGATTAAGGTGGACACAG (Coh3)

R-AAGCTTCTAATCTCCAACATTTACTCCACCGTC (for both clones)

PCR products were purified and cleaved with restriction enzymes EcoRI and HindIII and inserted into the pMAL-c2x expression vector (New England Biolabs), which contains the MBP gene immediately upstream of the EcoRI and HindIII restriction sites. The resultant plasmids encode, respectively, Linker-Coh3 and Coh3 fused to the C-terminal residue of MBP.

2.1.1 Plasmids and yeast strains

DNA fragments encoding each of the known or predicted subunits of a protein complex and their deletion or mutant derivatives are cloned into the two-hybrid expression vectors pGBT9 (TRP1) and pGAD424 (LEU2) (Bartel et al., 1993) or their commercially available equivalents (Clontech, Stratagene, etc.), fusing the yeast proteins to the Gal4 (or appropriate transcription factor) DNA-binding domain (DBD) and activation domains (AD), respectively. We recommend introducing an NdeI site at the 5′ end of the ORF (in which the ATG triplet of the recognition site sequence 5′-CAT ATG-3′ corresponds to the start codon of the ORF) following the site used to clone the DNA fragment, if the NdeI site is not present in the ORF. Then the NdeI site is used to move the DNA segment to GST fusion or yeast expression vectors (see below).

Y190 (α leu2 trp1GAL-lacZ GAL-HIS3) and Y187 (a leu2 trp1GAL-lacZ) (Harper et al., 1993), which contain chromosomal copies of the lacZ and HIS3 genes under the regulation of the upstream activation sequence (UAS) recognized by Gal4p, were transformed with the resulting pGBT9- and pGAD424-derived plasmids, respectively, as described (Schiestl et al., 1993).

Separation

DNA fragments are difficult to separate under normal CE conditions due to their virtually constant charge-to-mass ratio. Therefore, analyses are performed using a replaceable sieving matrix, consisting of a water-soluble polymer dissolved in a suitable buffer. Such solutions are referred to as entangled polymer buffers and the DNA sieved based on its ability to fit within pores created within the polymer matrix. The fact that these polymer matrices are not rigid makes them different from rigid agarose or polyacrylamide gels traditionally used in DNA analysis. The advantage of using an entangled polymer buffer is that fresh polymer solution can be pumped into the capillary at the conclusion of each analysis, cleaning the capillary and limiting problems with carryover. Experiments carried out using a variety of entangled polymer buffers have shown that with careful optimization of molecular weight and concentration, high-resolution DNA separations can be produced.

Several different mechanisms have been postulated to describe the separation of DNA in physical gels. These include transient entanglement coupling, Ogston sieving, and reptation. At low concentrations of polymer, separation takes place by means of a frictional interaction between the DNA and the polymer strands. This mechanism is known as transient entanglement coupling. At higher concentrations of polymer, individual polymer molecule strands begin to interact, producing a mesh. The polymer concentration at which this occurs is known as the entanglement threshold. Above the entanglement threshold, DNA fragments separate by sieving through transient pores created in the polymer mesh (Figure 1). Fragments, which are larger than the average pore size, reptate or move in a snake-like manner through the mesh. The key to producing an acceptable separation is to specify a polymer concentration at which the size of these virtual pores approximates the radius of gyration of the DNA fragment (average size of a DNA fragment in solution).

There are a number of key parameters involved in the development of a reliable separation of DNA using entangled polymers. In addition to concentration, the polymer length must be kept to a minimum to reduce viscosity and permit refilling of the capillary. Other important characteristics of entangled polymers include the relative stiffness and polydispersity of the polymer and its ability to coat the capillary walls. Uncoated silica capillaries have significant wall charge at the pH used to separate DNA. These charges can induce a bulk flow in the capillary walls when the electric fields are high. This effect is known as the electroosmotic flow (EOF) and can result in irreproducible changes in DNA migration from run to run. EOF is minimized by using polymers such as poly dimethyl acrylamide (POP), which coat capillary walls and neutralize wall charge effects. Furthermore, internal dye-labeled ladder standards are added to help compensate for any mobility shifts during the run.

Another important issue in DNA separation is the flexibility of the molecule, which can be characterized by a parameter known as the persistence length. ssDNA is far more flexible (shorter persistence length) and produces superior separations when compared to dsDNA, which is quite stiff and interacts poorly with the polymer matrix. As a result, it is very important to denature the DNA, and maintain it in its single-stranded state throughout the separation. To do this, the DNA is denatured in formamide before injection, and separations are carried out at elevated temperatures and with high concentrations of denaturants, such as urea and pyrrolidinone, to maintain this denatured state. Generally speaking, dsDNA migrates faster and at lower resolution in standard CE systems. Its appearance can sometimes be observed in improperly denatured samples.

Hybrid Capture

DNA fragments are hybridized from a whole-genome library to complementary sequences that have been synthesized and combined into a mixture of probes designed with high specificity for the matching regions in the genome. Covalently linked biotin moieties enable a secondary capture by mixing the probe library complexes with streptavidin-coated magnetic beads. The targeted regions of the genome are selectively captured from solution by applying a magnetic field, whereas most of the remainder of the genome is washed away in the supernatant. Subsequent denaturation releases the captured library fragments from the beads into solution, ready for postcapture amplification, quantitation, and sequencing. Exome sequencing is performed when the probes are designed to capture essentially all of the known coding exons in a genome [143].

The use of NGS has allowed different international initiatives such as The Cancer Genome Atlas (TCGA) or the International Cancer Genome Consortium (ICGC) to define the genomic landscape of early-stage breast cancer.

6 Step 1: Cell-Free Transcription–Translation and Fluorescence Monitoring

6.4 Tip

To measure kinetics under other buffer conditions, such as reduced magnesium concentration, the composition of Solution I of the PUREfrex kit, which contains all the low-molecular components, can be modified according to a previously reported method (Shimizu et al., 2001; Fig. 3).

Figure 3. Flowchart of Step 1.

Southern Blot Method

Southern Blot Application

Southern blotting has numerous research and clinical applications. For example, the technique is used in the clinical molecular diagnosis of myotonic dystrophy. The disorder is caused by abnormal expansion of a region of CTG trinucleotide repeats in the DMPK gene. Unaffected individuals have 5–35 copies of the CTG repeat, while affected individuals can have several thousand copies. A normal or slightly enlarged number of repeats is detected with PCR. The number of repeats in an allele with a large expansion is determined by the size of bands on a Southern blot (Figure 1).

Figure 1. Southern blot application to the diagnosis of myotonic dystrophy type 1. BglI restriction endonuclease-digested genomic DNA of affected and unaffected individuals, hybridized with fluorescein-labeled probe (sequence adjacent to the CTG repeat) and detected with the anti-FITC–AP reaction to a chemiluminescent substrate. Lane (1) unaffected individual homozygous for five CTG repeats; lane (2) individual affected with myotonic dystrophy type 1 with a normal allele of five repeats and an abnormal allele of 130 CTG repeats; and lane (3) individual affected with myotonic dystrophy type 1 with a normal allele of five repeats and an abnormal allele of 700 repeats.

6 Step 2 End-Repair the Fragmented DNA

6.5 Tip

This is a stopping point and purified DNA can be stored at 4 °C, or can be taken directly for Adaptor Ligation.

See Fig. 15.3 for the flowchart of Step 2.

Figure 15.3. Flowchart of Step 2.

IV. Effect of Mutations in Polycomb Group Genes on mini-white Silencing

If mini-white silencing is due to repression by PcG proteins, then removal of those proteins should lead to an increase in eye color. Because mutations in most Polycomb group genes are homozygous lethal, the effects of removal of one copy on either mini-white variegation in heterozygotes or on pairing-sensitive silencing in homozygotes has been examined. Mini-white silencing caused by some DNA fragments was suppressed by heterozygous mutations in PcG genes (Table 14.1). In these instances, mini-white silencing was suppressed at many, but not all, chromosomal insertion sites (Hagstrom et al., 1997; Muller et al., 1999; Tillib et al., 1999; Horard et al., 2000). For other DNA fragments, heterozygous mutations in PcG genes had no effect or only affected a small number of chromosomal insertion sites (Kassis, 1994; Kapoun and Kaufman, 1995a; Gindhardt and Kaufman, 1995). For the polyhomeotic DNA-induced variegation, the results are puzzling. While polyhomeotic mutations suppressed both variegation and PS silencing as expected; Polycomb mutations enhanced silencing in heterozygotes but had no effect on pairing-sensitive silencing (Fauvarque and Dura, 1993). These results are puzzling because Polycomb and polyhomeotic are both thought to act as transcriptional repressors and may act in the same protein complex (Shao et al., 1999).

Since there are different types of PcG protein complexes, there might be different PREs for the recruitment of each complex. Thus, we might be able to identify DNA fragments that render mini-white silencing sensitive to only a subset of PcG genes. In general, this has not been observed (note one exception in Tillib et al., 1999). Instead, the chromosomal insertion site seems to dictate which PcG mutations effect silencing (Kassis, 1994; Hagstrom et al., 1997; Muller et al., 1999; Horard et al., 2000). This suggests that the silencing of mini-white at a particular chromosomal location is the result of an interaction between proteins bound to flanking genomic PREs and the PREs present in the transposon (as proposed in Pirrotta, 1997a, 1997b). Since mini-white silencing at particular chromosomal insertion sites can be sensitive to mutations in only a subset of PcG genes, it seems that not all PcG proteins contribute to silencing of all PREs. However, the extreme position dependence of PRE silencing in transgenes, along with the clustering of PS sites within PREs suggests that PREs do act independently.