DNA Technology & Genomics CHAPTER 20

Restriction Enzymes enzymes that cut DNA at specific locations (restriction sites) yielding restriction fragments used to insert genes into bacterial plasmids – the sticky ends of the restriction fragments can base-pair with the sticky ends of a cut plasmid & then be sealed together by DNA ligase

Genomic Libraries the process of DNA cloning usually results in thousands of different types of recombinant plasmids – the complete set of these recombinant plasmids is called a genomic library one method for identifying bacteria with the recombinant plasmid containing the gene of interest is nucleic acid probe hybridization *white colonies

Nucleic Acid Hybridization

Expressing Eukaryotic Genes in Prokaryotic Cells Problem 1 – bacterial cell doesn’t recognize the eukaryotic gene’s promoter – solution: link the gene of interest to a highly active prokaryotic promoter that the bacterial cell will recognize this is accomplished by inserting the gene of interest into a cloning vector (plasmid) that contains this promoter Problem 2 – eukaryotic genes contain introns & bacterial cells lack RNA-splicing machinery – solution: insert the cDNA (complimentary DNA) form of the gene

Creating cDNA mRNA transcript of gene of interest is combined with reverse transcriptase to create a complimentary DNA strand the mRNA strand is then degraded and the complimentary, second strand of DNA is created by adding DNA polymerase the resulting cDNA molecule is modified by adding sticky ends so that it can be inserted into a cloning vector

Eukaryotic Hosts for DNA Cloning yeast cells (which also contain plasmids) can be used as hosts for DNA cloning to avoid eukaryotic-prokaryotic incompatibility

PCR (Polymerase Chain Reaction) alternative method for cloning a gene used primarily when the source of DNA is scanty or impure applications: – amplify fragments of ancient DNA – amplify DNA from fingerprints or tiny amounts of blood, tissue, or semen found at crime scenes – amplify DNA from embryonic cells to test for genetic disorders

Restriction Fragment Analysis the DNA fragments produced by restriction enzyme digestion can be sorted by gel electrophoresis: – uses a gel & an electric field to separate nucleic acids or proteins on the basis of size and charge the bands produced by gel electrophoresis can be analyzed for a particular gene using nucleic acid hybridization (this technique is called Southern blotting)

GENOME MAPPING 3 stages : linkage mapping physical mapping DNA sequencing

Cytogenetic Maps the starting point for DNA mapping shows a chromosome’s banding pattern and the location of specific genes on the chromosome – the genes were located using a technique called FISH (fluorescence in situ hybridization) in which fluorescently labeled probes are allowed to hybridize to an immobilized array of whole chromosomes

Linkage Maps a map of genetic markers based on recombination frequencies the markers can be: – genes – RFLPs (restriction fragment length polymorphisms) – different restriction fragment patterns on homologous chromosomes due to differences in their restriction sites – simple sequence DNA – sections of DNA that contain repeated short sequences

Physical Maps express the distance (number of base-pairs) between markers made by cutting the DNA of a chromosome into a number of restriction fragments and then determining the original order of the fragments in the chromosome by looking for areas where the fragments overlap

DNA Sequencing determining the complete nucleotide sequence of each chromosome techniques for DNA sequencing include: – dideoxy chain-termination method – shotgun approach developed by Celera Genomics skips the linkage & physical mapping stages takes random DNA fragments & uses computer programs to sequence and order them

shotgun approach dideoxy chain- termination method

Human Genome Project sequencing of the 22 autosomes and the sex chromosomes (largely completed in 2003) also involves mapping the genomes of other species important to biological research allows scientists to study whole sets of genes & their interactions

Searching for Protein-Coding Genes scientists used software to scan DNA sequences for: – transcriptional & translational start and stop sequences – RNA-splicing sites – short coding sequences (expressed sequence tags) similar to those in known genes

Determining Gene Function disable gene and observe the consequences – in vitro mutagenesis – a mutation is introduced in a cloned gene & then gene is returned to the cell – RNA interference (RNAi) – synthetic, double- stranded RNA molecules matching the sequence of a particular gene are used to trigger the breakdown or block the translation of the gene’s mRNA

Studying Gene Expression to determine which genes are expressed in a particular cell, scientists can use DNA microarray assays – isolate all the mRNAs made in a particular cell – use them to make fluorescently-labeled cDNAs by reverse transcription – apply the cDNA to a microarray containing all the organism’s genes

Comparing Genomes of Different Species allows scientists to determine evolutionary relationships between species helps scientists better understand the human genome provides scientists with a scaffold for organizing the DNA sequences of closely related species makes it easier for scientists to correlate phenotypic differences between species with particular genetic differences can be used to help study certain genetic diseases

Future Areas of Research Proteomics – studying the full protein sets encoded by genomes studying human evolution & the history of human populations through the identification of SNP sites – SNPs (single nucleotide polymorphisms) are single base-pair variations that account for the genetic differences between individuals (which is only about 0.1%)

Medical Applications diagnosing diseases detecting susceptibility of diseases treating diseases – gene therapy – alteration of an afflicted individual’s genes APPLICATIONS OF DNA TECHNOLOGY

Pharmaceutical Applications manufacture large quantities of human proteins (ex: insulin) production of vaccines pharmacogenomics

Forensic Applications DNA fingerprinting

Environmental Cleanup genetically engineered microbes are being used or created to… – remove heavy metals from the environment – cleanup highly toxic mining wastes – degrade chlorinated hydrocarbons in wastewater – breakdown chemical released during oil spills – breakdown toxic wastes in waste dumps

Agricultural Applications make vaccines & growth hormones for treating farm animals creating transgenic animals & plants