Directional cDNA Cloning

The synthesis of cDNA using modified oligonucleotides to initiate each strand of DNA synthesis allows the insertion of unique restriction enzyme recognition sites at either end of the cDNA so that cloning of the cDNA fragments can only occur in one direction (Figure 5.6). In the example shown here, the oligo-dT primer also contains additional sequences at the 5'-end that encode a XhoI 5'-l IAAAAA- 3' 3'-TTTTTGAGCTCGGG-5' I Reverse transcriptase Alkaline sucrose gradient Primer -...

Chloroplast Transformation

In recent years, chloroplast transformation has emerged as a serious alternative to nuclear transformation (Figure 11.5). The chloroplast, like the mitochondrion, possesses its own genome. The chloroplast genome is a single circular double-stranded DNA molecule, which, in the model dicotyledonous plant Ara-bidopsis thaliana, is composed of approximately 155 kbp of DNA containing 87 protein coding genes (Sato et al., 1999). Chloroplast genes, like those of bacteria, are generally arranged into...

Cosmid Vectors

The only DNA requirements for in vitro packaging into X phage are the presence of two cos sites that are separated by 37-51 kbp of intervening sequence. Cosmids were developed in light of this observation, and are simply plasmids that contain a X phage cos site (Collins and Briining, 1978). Figure 3.14 shows the overall architecture of a cosmid vector and a cloning scheme for the insertion of foreign DNA. As plasmids, cosmids contain an origin of replication and a selectable marker. Cosmids...

The GAL System

Gal Promoter

In yeast, like almost all other cells, galactose is converted to glucose-6-phosphate by the enzymes of the Leloir pathway. Each of the Leloir pathway structural genes collectively called the GAL genes are expressed at a high level, representing 0.5-1 per cent of the total cellular mRNA St John and Davis, 1981 , but only when the cells are grown on galactose as the sole carbon source. Each of the GAL genes contains within its promoter at least one, and often multiple, binding sites for the...

The T7 Expression System

Iptg Induction Polymerase

This is the RNA polymerase encoded by bacteriophage T7 is different from its E. coli counterpart. Unlike the a2 2 subunit structure of the E. coli enzyme, T7 RNA polymerase is a single-subunit enzyme that binds to distinct DNA 17 bp promoter sequences 5'-TAATACGACTCACTATA-3' found upstream of the T7 viral gene it activates. E. coli RNA polymerase does not recognize T7 promoter sequences as start sites for transcription. The overall scheme for the production of target proteins using the T7...

DNA Purification

Cscl Etbr Gradient Supercoiled Dna

Many of the techniques we have discussed in this chapter are heavily reliant upon the availability of relatively large quantities of purified DNA. The isolation of nuclear material from cells is a relatively straightforward process. Lysis of Proteins separated through polyacrylamide gel Block non-specific protein binding sites and add primary antibody Wash away excess primary and add labelled secondary antibody Wash away excess primary and add labelled secondary antibody Figure 2.23. Western...

Ccc Aac Cac Atc Cgc Cac Aag Atc Gtg Gag Atg Gcc Cac Cac Ggc Atc Cgg Ccc Tgc P N H I R H K I V E M A H H G I R P C

GTC ATC TCG CGC CAG CTG CGC GTG TCC CAC GGC TGC GTC TCC AAG ATC CTG-3' V I S R Q L R V S H G C V S K I L 3'-AGG GTG CCG ACG CAG AGG TTC TAG-5' Primer 2 Figure 4.9. PCR to detect a pathological deletion mutation. A. The wild-type DNA sequence of the sense strand of part of exon 2 from the human Pax-3 gene. The sequence shown represents the first part of the paired box, a highly conserved element that functions as a transcription factor. Certain patients suffering from Waardenburg syndrome...

Phosphorothioate Strand Selection

A phosphorothioate nucleotide contains a phosphorus-sulphur linkage in place of a phosphorus-oxygen group Figure 7.3 . If phosphorothioate deoxynu-cleotides in which the sulphur is attached to the a-phosphate are used in a DNA synthesis reaction, then the phosphorothioate will be incorporated into the newly synthesized DNA. Certain restriction enzymes are unable to cleave DNA that contains phosphorothioates Nakamaye and Eckstein, 1986 . The mutagenic oligonucleotide is annealed to the...

M13 Vectors

Dna Replication M13

M13, and its very close relatives f1 and fd, are filamentous E. coli bacterio-phages. M13 is a male-specific lysogenic phage with a circular single-stranded DNA genome 6407 bp in length Figure 3.15 . M13 phage particles have dimensions of about 900 nm x 9 nm and contain a single-stranded circular DNA molecule designated as the strand . M13 infects bacteria that harbour the F pilus. The phage particle absorbs via one end to the F pilus, and the single-stranded phage DNA enters the bacterium...

Restriction Enzyme

Dna Ligase Resteriction Enzyme

The mechanism of DNA joining by DNA ligase. See the text for details. This figure is adapted from Doherty et al. 1996 The ability to break and rejoin DNA molecules almost at will led to the first experiments in DNA cloning in 1972 Jackson, Symons and Berg, 1972 . Figure 2.7. Breaking and joining DNA using restriction enzymes and DNA ligase. Linear DNA insert and a closed-circular plasmid DNA vector each contain the recognition site for BamHI and EcoRI. Mixing the DNA fragments with...

Pronuclear Injection

Dna Pronuclear Injection

As with the methods we have previously discussed for the direct injection of DNA fragments into Xenopus oocytes Chapter 12 , DNA can be injected directly into the pronuclei of freshly fertilized mouse eggs Palmiter and Brinster, 1986 . Immediately following fertilization, the large male and small female pronuclei are visible under the microscope as discrete entities. DNA injections are usually made into the larger male pronucleus while the egg is being held in position using a suction pipette...

The Restriction Enzyme Ecorirecognizes And Cleaves

Hhal Methylase

Protection of DNA cleavage by modification. The restriction enzyme EcoRI recognizes and cleaves the sequence 5 -GAATTC-3'. E. coli R strains, from which this enzyme is derived, protect their own DNA from fragmentation by also producing a specific methylase. EcoRI methylase takes a methyl group from S-adenosylmethionine and places it on the N6 of the second adenine in the recognition sequence. EcoRI restriction enzyme is unable to cleave the methylated DNA semiconservative DNA...

Images Of Zig Zag Bands From Pulse Field Gel Electrophoresis

Dna Fragment Size Distance Migrated

The migration of DNA fragments through an agarose gel. a An agarose gel showing the separation of DNA fragments of known size. b The size of a DNA fragment and the distance migrated through the gel. c A plot of fragment size against distance migrated taken from the data shown in b . This indicates that the relationship between fragment size and distance migrated is not linear. d A plot of the log of fragment size against distance migrated. This indicates that there is a direct,...

Genomic Libraries

The smallest unit of DNA within a genome is the chromosome. Even in the simplest organisms, however, chromosomes contain an enormous quantity of DNA. For example, the E. coli chromosome contains some 4.6 Mbp 4 600 000 bp of DNA Table 5.1 . This amount of DNA is far too large to be cloned into any of the vectors currently available Chapter 3 . Therefore it is necessary, and indeed desirable, to fragment the DNA before it is cloned into an appropriate vector. A 'divide and conquer' strategy comes...

The Eukaryotic Nucleosome

Protein Scaffold Nucleosomes

Each cell within our body contains a huge amount of DNA. The different chromosomes of the human genome contain approximately 3.2 x 109 base pairs of DNA. Since we are diploid organisms, having two sets of each chromosome, the total amount of DNA in most of our cells totals 6.4 x 109 base pairs. At 0.33 nm per base pair Figure 1.7 , this corresponds to an overall length of approximately 2.1 m. How can this fit into a nucleus measuring just 5-10 m across The answer is that the DNA is highly...