Recombinant DNA Technology Chapter 14 Recombinant DNA Technology

Some terminology genetic engineering recombinant DNA technology deliberate modification of organism’s genetic information by directly changing the sequence of nucleic acids in its genome recombinant DNA technology procedures used to carry out genetic engineering biotechnology use of organisms to form useful products

Restriction enzymes bind to DNA at specific sequence called the recognition site cleave DNA at this site or a defined distance from it hundreds are commercially available Figure 14.2

Restriction Endonucleases and Their Recognition Sequences Table 14.2

Staggered cuts Sticky ends Cohesive ends ligase

Reverse transcriptase synthesizes double stranded DNA from RNA template used to construct complementary DNA (cDNA) independently discovered by Temin and Baltimore in 1970

cDNA Complementary DNA Eukaryotic genes – introns and exons Introns – noncoding regions Exons - coding regions cDNA – synthetic gene – has only exons c stands for complementary DNA

If we want to introduce eukaryotic gene into a prokaryotic cell, we should use cDNA If we place natural eukaryotic gene into a Bacterial cell, it cannot remove the introns. Functional protein will not be produced by the prokaryotic cell.

Southern blotting technique developed by Edwin M. Southern (1975) used to detect specific DNA fragments often uses radioactive DNA hybridization probes autoradiography method for detecting radioactively labeled molecules

Genetic screening. If a person is a carrier for cystic fibrosis (genetic disorder) Mutation in a gene that codes for a membrane protein Thick mucus

Person has the disease – symptoms Some people are carriers One cystic fibrosis gene (father) one normal gene (mother) – heterozygous for cystic fibrosis Two carriers – have a baby – the baby can have the disease Genetic screening is used – procedure known as southern blotting.

Gel electrophoresis hybridize Single stranded DNA complementary

Synthetic DNA oligonucleotides (can be DNA or RNA) synthesized in stepwise process in which single nucleotides are added to end of a growing DNA chain chain length can vary from short (2-30 nucleotides) to long (50-100 nucleotides) can be synthesized to have known nucleotide sequences DNA synthesizer – products are used in PCR

The Polymerase Chain Reaction (PCR) Amplify enables the rapid synthesis of many copies of a specific DNA fragment from a complex mixture of DNA and other cellular components reaction mix contains primers target DNA thermostable DNA polymerase such as Taq polymerase each of the four deoxyribonucleotide triphosphates thermocycler is the instrument used

Polymerase Chain Reaction Amplify DNA Target DNA Nucleotides DNA polymerase

More About PCR… in the reaction DNA is denatured primers anneal to target DNA copies of the target DNA are synthesized

Cloning Cellular DNA Fragments Figure 14.10

Gel Electrophoresis used to separate molecules based on their charge and size agarose or acrylamide gels can be used to separate DNA fragments DNA is acidic; it migrates from the negative to the positive end of the gel each fragment’s migration rate is inversely proportional to the log of its molecular weight

Gel Electrophoresis of DNA Figure 14.11

Cloning Vectors and Creating Recombinant DNA there are four types of cloning vectors plasmids (most commonly used) phages and viruses cosmids artificial chromosomes

Recombinant DNA Cloning Vectors Table 14.3

Cloning Vectors and Creating Recombinant DNA each type of cloning vector generally has an origin of replication a selectable marker a multicloning site or polylinker

Vectors – carry the gene of interest into a bacterial cell. Plasmids Small enough – they can enter into the cell Selection markers – antibiotic resistance genes. Help in selecting the cells that have the gene of interest.

Construction of Genomic Libraries used when gene of interest is on a chromosome that has not been sequenced the library is constructed by cleaving the genome and then cloning the fragments into vectors the libraries are screened for the genes of interest in a variety of ways

Gene library Collection of genes from an organism Mouse gene library, yeast gene library Biotechnology company Yeast gene library DNA – fragment – restriction enzyme – fragments are inserted into plasmids – introduced into a bacterial cell Each bacterial cell having the recombinant plasmid is a clone Large # clones – a clone for each gene that exists in the yeast cell

Hexokinase Malate Dehydrogenase pheromone Helps the cells To come together And mate

Inserting Recombinant DNA into Host Cells most common hosts E. coli - procaryotic host S.cerevisiae – eucaryotic host DNA introduction into microbes transformation

The Numerous Applications of Genetic Engineering in medicine many useful proteins gene therapy - mutant gene replaced by normal gene. in agriculture

Table 14.4

Agriculture

Forensic medicine Each person has a unique set of introns. restriction enzyme fragment pattern of DNA

Social Impact of Recombinant DNA technology there is scientific and philosophical concern about the use of embryonic stem cells in gene therapy. This is currently a major controversial issue in the U.S. the production of genetically engineered food the possibility of the production of genetically engineered organisms by bioterrorists and other issues