DNA TECHNOLOGY DNA recombination or genetic engineering is the direct manipulation of genes for practical purposes

Recombinant DNA technology Refers to the set of techniques for combining genes from different sources in vitro( in a test tube ) and transfering this DNA into a cell so it can be expressed. These techniques were first developed around 1975 and resulted in the appearance of the Biotechnology industry

12.7 DNA technology is changing the pharmaceutical industry –DNA technology Is widely used to produce medicines and to diagnose diseases CONNECTION

What is biotechnology? The use of living organisms to do practical tasks. Examples: The use of microorganisms to make cheese and wine Selective breeding of livestock and crops Production of antibiotics from microorganisms Production of monoclonal antibodies

What is the goal of biotechnology? To find practical applications of DNA tecniques for the improvement of human health and food production Examples: Making gene products using Genetic Engineering Uses in basic research Medical uses. Diagnosis of disease Making vaccines and other pharmaceutical products Forensic uses of DNA such as DNA fingerprinting Agricultural uses such as manipulatingplant genes and making transgenic plants.

The tools of recombinant DNA Plasmids Restriction enzymes Gel electrophoresis PCR ( polymerase chain reaction)

BACTERIAL PLASMIDS AND GENE CLONING 12.1 Plasmids are used to customize bacteria: An overview –Gene cloning is one application of DNA technology Methods for studying and manipulating genetic material

–Researchers can insert desired genes into plasmids, creating recombinant DNA And insert those plasmids into bacteria Bacterium Bacterial chromosome Plasmid 1 isolated 3 Gene inserted into plasmid 2 DNA isolated Cell containing gene of interest DNA Gene of interest Recombinant DNA (plasmid) 4 Plasmid put into bacterial cell Recombinant bacterium 5 Cell multiplies with gene of interest Copies of protein Copies of gene Clone of cells Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein used to dissolve blood clots in heart attack therapy Protein used to make snow form at higher temperature Figure 12.1

Therapeutic hormones –In 1982, humulin, human insulin produced by bacteria Became the first recombinant drug approved by the Food and Drug Administration Figure 12.7A

–Gene therapy Is the alteration of an afflicted individual’s genes Gene therapy may someday help treat a variety of diseases Cloned gene (normal allele) 1 Insert normal gene into virus 2 Infect bone marrow cell with virus 3 Viral DNA inserts into chromosome 4 Inject cells into patient Bone marrow Bone marrow cell from patient Viral nucleic acid Retrovirus Figure 12.13

12.11 Restriction fragment length polymorphisms can be used to detect differences in DNA sequences

How Restriction Fragments Reflect DNA Sequence –Restriction fragment length polymorphisms (RFLPs) Reflect differences in the sequences of DNA samples Crime sceneSuspect w x yy z Cut DNA from chromosomes C C G G G G C C A C G G T G C C C C G G G G C C C C G G G G C C Figure 12.11A

12.9 DNA microarrays test for the expression of many genes at once –DNA microarray assays Can reveal patterns of gene expression in different kinds of cells CONNECTION

–DNA microarray 1 mRNA isolated Reverse transcriptase and fluorescent DNA nucleotides 2 cDNA made from mRNA 4Unbound cDNA rinsed away 3 cDNA applied to wells DNA microarray Each well contains DNA from a particular gene Actual size (6,400 genes) Nonfluorescent spot Fluorescent spot cDNA DNA of an expressed gene DNA of an unexpressed gene Figure 12.9

12.10 Gel electrophoresis sorts DNA molecules by size ++ – – Power source Gel Mixture of DNA molecules of different sizes Longer molecules Shorter molecules Completed gel Figure 12.10

–After digestion by restriction enzymes The fragments are run through a gel – + Longer fragments Shorter fragments x w y z y 12 Figure 12.11B

12.11 Restriction fragment length polymorphisms can be used to detect differences in DNA sequences

Using DNA Probes to Detect Harmful Alleles –Radioactive probes Can reveal DNA bands of interest on a gel

–Detecting a harmful allele using restriction fragment analysis Restriction fragment preparation Gel electrophoresis Blotting Radioactive probe Detection of radioactivity (autoradiography) IIIIII IIIIII Restriction fragments Filter paper Probe Radioactive, single- stranded DNA (probe) Film I II III I II III Figure 12.11C

DNA technology is used in courts of law CONNECTION

DNA and Crime Scene Investigations –Many violent crimes go unsolved For lack of enough evidence –If biological fluids are left at a crime scene DNA can be isolated from them

–DNA fingerprinting is a set of laboratory procedures That determines with near certainty whether two samples of DNA are from the same individual That has provided a powerful tool for crime scene investigators Investigator at one of the crime scenes (above), Narborough, England (left)

DNA Fingerprinting 1 st - The DNA molecule is cut with restriction enzymes 2 nd - we have to separate the fragments This is done by a technique called gel electrophoresis The DNA is placed on a tray filled with gel through which an electric current runs causing the fragments to move through the gel. The segments separate by how far they move in the gel according to size. The DNA will form bands corresponding to the bases (and no two people have the same sequence of bases) in the gel which are unique for each individual. This is DNA fingerprinting

–DNA fingerprinting can help solve crimes Defendant’s blood Blood from defendant’s clothes Victim’s blood Figure 12.12A Figure 12.12B

Safety and ethical issues

Methods for purifying the DNA Vectors for carrying the DNA into cells and replicating it Techniques for determining nucleotide sequences of DNA molecules