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Molecular Techniques in Radiobiology Introduction The Introduction • The birth of molecular biology could be ascribed to the discovery of the structure of DNA by Watson Molecular Techniques in and Francis Crick in 1953 Radiobiology • In the late 1940s and early 1950s, Linus Pauling realized that as amino acids were folded into proteins, they formed three-dimensional structures, and that function was related to the structure th Chapter 16 (6 edition) • The whole concept emerged that the sequence of bases, which coded for a protein, ultimately Eric J. Hall., Amato Giaccia, determined function leading the way to breaking the genetic code Radiobiology for the Radiologist The structure of DNA The structure of DNA • DNA - deoxyribonucleic acid is a polymer with the • DNA molecule has many monomer units of nucleotides deoxyribo-nucleotides (bases) linked in a chain- • Each nucleotide consists of a 5-carbon sugar like arrangement (deoxyribose), a nitrogen containing base attached to the sugar, and a phosphate group • Bases are held by hydrogen bonds and are • There are four different types of nucleotides found in paired complimentary: DNA, differing only in – adenine with thymine the nitrogenous base: – cytosine with guanine A is for adenine • Each half constitutes a G is for guanine template for C is for cytosine reconstruction of the T is for thymine other half RNA and DNA Transcription and translation • RNA – ribonucleic acid, has ribose sugar molecule The flow of genetic information from DNA to protein (gene instead of deoxyribose expression) requires a series of steps: • In the cell RNA is usually single-stranded, while • In the first step, the DNA code is transcribed in the nucleus into mRNA (messanger RNA); transcription is controlled by other DNA is usually double-stranded DNA sequences (such as promoters), which show a cell where • RNA has the base uracil rather than thymine that genes are, and control how often they are copied is present in DNA • During the second step, the RNA copy made from a gene is then • RNA has a much shorter chain of nucleotides fed through a ribosome, which translates the sequence of • Unlike DNA, which is located primarily in the nucleotides (with help of ribosomal RNA and transfer tRNA) in nucleus, RNA is found throughout the cell the mRNA into the correct sequence of amino acids and joins these amino acids together to make a complete protein chain • There are several types of RNA • The new protein then folds up into its active form 1 Transcription and translation Amino acids and proteins • Introns and exons are nucleotide sequences within genes • Each amino acid is specified by triplet •Typically introns make mRNA sequence, codon; four bases give a up the bulk of the gene possibility to have 4x4x4=64 combinations • Introns are removed during the process of • There are only 20 amino acids; more than splicing one triplet can code each amino acid • Remaining exons are joint together • The vast majority of proteins is composed • Only the exons of the of a mixture of the same 20 amino acids; DNA are translated chains vary from 5 to > 4000 amino acids Gene manipulation tools Gene manipulation tools • The most common form of genetic • Restriction endonucleases: enzymes found in manipulation (engineering) involves the bacteria that can recognize specific DNA sequence insertion of new genetic material in the host and cleave at or near that site genome at a random or specific location • Recognition sites are short, 4 to 8 nucleotides, and usually read the same in both directions • Need to have abilities to recognize specific (palindromic sequences) sequences, cleave DNA, replicate the • There are types I,II, and III, but only type II cut pieces, insert it into a host the DNA without modifications, and are used in molecular biology Gene manipulation tools Gene manipulation tools • Some endonucleases produce blunt-ended fragments because they cut cleanly through the • A vector is a self-replicating DNA molecule that DNA, cleaving both has the ability to carry a foreign DNA molecule complementary strands at the into a host cell same nucleotide position, most often near the middle of the • Vectors are typically used to insert fragments of recognition sequence human DNA, containing a gene of interest, into a • Other endonucleases cleave the bacterium that will self-replicate, producing two strands of DNA at positions quantities enough for a study two to four nucleotides apart, • There are many types of vectors: plasmids, creating exposed “sticky” ends bacteriophages, bacterial artificial chromosomes of single-stranded sequences, very useful for making (BACs), and viruses recombinant molecules 2 Gene manipulation tools Gene manipulation tools • Bacteriophage can infect its host at a much higher efficiency than a plasmid • It can accommodate a larger range of DNA • Plasmids contain genes for resistance to bacteriophages, antibiotics, fragments, from a few to and some heavy metals up to 24,000 bp • Only those bacteria that have taken up the plasmid survive and replicate in a culture medium containing the antibiotic • Useful only for relatively small DNA inserts (~10,000 bps) • Do not transfect into bacteria with high efficiency Gene manipulation tools Gene manipulation tools: hosts • BACs (bacterial artificial chromosomes) are vectors based on a type of plasmid with sequences encoding self-replication. BACs can accommodate approximately 300 kilobases (kb) of • Recombinant DNA molecules can be DNA (plasmids are limited to approximately 10-kb insertions) constructed and manipulated to some extent • BACs were the primary vector used during the genome- sequencing projects, mainly because a BAC carrying a gene of in the test tube, but amplification and interest is easily acquired expression ideally require a host: • Viruses are highly efficient vectors for introducing foreign genes into mammalian cells – Escherichia coli – Retroviruses are a type of RNA virus. The RNA genome of – Yeast retroviruses is transcribed into DNA, which is then integrated into the host genome. Retroviruses can infect virtually every type of – Mammalian cells with limited or unlimited mammalian cell, making them very versatile. – Adenoviruses are a type of DNA virus lifespan Gene manipulation tools: hosts Agarose gel electrophoresis • Mammalian cells do not take up foreign DNA naturally; one of several tricks must be used to bypass natural barriers • DNA is negatively charged – Microinjection: DNA can be injected, cell by cell, directly into the nucleus through a fine glass needle. • Can separate pieces of – Calcium phosphate precipitation: Cells take up DNA relatively efficiently DNA of different sizes in the form of a precipitate with calcium phosphate – Cationic lipids: offer some of the highest transfection efficiencies and • After separation is expression levels to a wide variety of cells, both in suspension and complete ethidium attached – Electroporation: Cells in solution are subjected to a brief electrical pulse bromide is added, making that causes holes to open transiently in the membrane, allowing foreign DNA fluoresce under DNA to enter ultraviolet light to make – Viral vectors: The ultimate means of transfection involves the use of a retrovirus. This is analogous to using bacteriophage to get DNA the position of the DNA into bacteria. Oncogenes, genes that can cause cancer, and their counterpart, tumor-suppressor genes, can be studied by incorporating visible them into retro-viral vectors 3 Polymerase chain reaction (PCR) Gene analysis: Southern blotting • DNA polymerase is an enzyme that catalyzes the polymerization of • Mapping technique, used to nucleotides into a DNA strand detect specific fragments of • Primers (short DNA fragments) DNA, named after British contain sequences complementary to biologist Edwin Southern the target region • Hybridization of the probe • The polymerase "reads" an intact DNA to a specific DNA fragment strand as a template and uses it to synthesize the new strand. This process on the filter membrane copies a piece of DNA indicates that this fragment • As PCR progresses, the DNA contains DNA sequence generated is itself used as a template that is complementary to the for replication, promoting a chain probe reaction • Can detect mutations, such as insertions, deletions and sequence differences in DNA Gene analysis: Chromosome walking Gene knockouts • Used when gene • Gene knockout technique involves making one of the has been mapped genes in a living organism inoperative to a specific arm • Used to learn about a gene that has been sequenced, of a chromosome, but has an unknown or incompletely known function but has not been isolated • Genes in embryonic stem cells are manipulated and • Probes are used to inserted into early identify an embryos overlapping • Gene function is inferred fragment of DNA from the difference from the genomic between the knockout library organism and normal A knockout mouse (left) that is a model of individuals obesity, compared with a normal mouse Other blotting techniques Databases and sequence analysis • Northern blotting - the technique for • GenBank, a database at the National Center for separating RNA by gel electrophoresis and is Biotechnology Information (www.ncbi.nlm.nih. analogous to the Southern blot technique used gov) for the deposition of individually cloned to study DNA genomic, cDNA and protein sequences • Related programs, called search engines, were • Western blotting – used to detect specific developed to find similar sequences from the ever- proteins in a homogenized tissue sample growing list of identified
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