DNA Technology and Genomics

DNA Technology Methods for studying and manipulating genetic material Cloning Genetically Modified Gene Therapy DNA Profiling Genomics

Recombinant DNA Combining nucleotide sequences from 2 sources to form a single DNA Bacteria often used Plasmid

Creating Recombinant DNA Restriction Enzymes Restirction site Sticky Ends Ligase 5

Cloning the Gene Bacteria containing the gene are cloned Typically, antibiotic resistance genes are also inserted Bacteria are grown on antibiotic medium All bacteria without the resistance (and target) gene die Only those with gene reproduce Stored in genomic library Plasmids used to “store” genetic information

Uses Gene Cloning Genetic Engineering Produces multiple copies of gene-carrying DNA Genetic Engineering Direct manipulation of genes

cDNA Express Eukaryotic genes in Prokaryotic cells mRNA What about introns? Prokaryotes don’t have machinery to splice mRNA Already spliced Work from mRNA to create cDNA

and addition of reverse transcriptase; synthesis of DNA strand Fig. 12-4 Cell nucleus Exon Intron Exon Intron Exon DNA of eukaryotic gene 1 Transcription RNA transcript 2 RNA splicing mRNA 3 Isolation of mRNA and addition of reverse transcriptase; synthesis of DNA strand Test tube Reverse transcriptase cDNA strand being synthesized 4 Breakdown of RNA 5 Synthesis of second DNA strand cDNA of gene (no introns)

Table 12.6

Uses Diagnosis and treatment of disease Vaccines Therapeutic hormones

Probes Genomic Library Nucleic Acid Probe How to find the right gene? Locate specific gene or nucleotide sequence

Probes Synthesize short sequence of singe stranded DNA of complimentary sequence Label radioactive tag Mix with genomic library Bacteria with gene of interest will glow

Radioactive DNA probe Mix with single- stranded DNA from Fig. 12-5 Radioactive DNA probe Mix with single- stranded DNA from genomic library Single-stranded DNA Base pairing indicates the gene of interest

Genetically Modified Organisms who have acquired genes by artificial means If from another species= transgenic organism Agrobacterium tumefaciens Plant cell DNA containing gene for desired trait 1 Ti plasmid Recombinant Ti plasmid 2 3 Introduction into plant cells Regeneration of plant Insertion of gene into plasmid DNA carrying new gene Plant with new trait Restriction site

Gene Therapy Altering an afflicted persons genes for therapeutic purposes Treatment of genetically based disorders Technology still in infancy

Cloned gene (normal allele) Insert normal gene into virus Fig. 12-10 Cloned gene (normal allele) 1 Insert normal gene into virus Viral nucleic acid Retrovirus 2 Infect bone marrow cell with virus 3 Viral DNA inserts into chromosome Bone marrow cell from patient Bone marrow 4 Inject cells into patient

DNA Profiling Forensics DNA Profiling Scientific analysis of evidence Analysis of DNA fragments Determine if they originate form a particular individual PCR Gel Electrophoresis

PCR 3 step cycle Heat separates strands Cooling allows primers to form H bonds with end of target sequence DNA Polymerase adds nucleotides Creates double stranded DNA Repeat

PCR Significant breakthrough in genetics Original polymerase was from E. coli Heating traditionally denatured polymerase New polymerase added after each heating cycle Giant pain in the butt

PCR Discovery of T. aquaticus Could now be done at higher temps Lives in hotsprings Not denatured under high temps Could now be done at higher temps Better success rates

PCR Cycle 1 yields 2 molecules Cycle 2 yields 4 molecules Cycle 3 Genomic DNA 3 5 3 5 3 5 5 1 Heat to separate DNA strands 2 Cool to allow primers to form hydrogen bonds with ends of target sequences 3 DNA polymerase adds nucleotides to the 3 end of each primer 3 5 5 3 Target sequence 5 5 3 5 3 5 3 Primer New DNA

Gel Electrophoresis Use of gel to separate DNA strands by size (molecular weight) or charge DNA must first be “digested” Strands must be cut into different sizes Use Restriction Enzymes Cut DNA in specific places Looks for specific nucletoide sequences

Gel Electrophoresis – + Mixture of DNA molecules of different sizes Power source Gel Mixture of DNA molecules of different sizes Longer molecules Shorter Completed gel Figure 12.10

Gel Electrophoresis STR Create a genetic profile of individuals Short sequences of DNA repeated many times in a row STR analysis compared lengths of STR sequences at specific sites on the genome Create a genetic profile of individuals STR’s differ among individuals

Gel Electrophoresis RFLP Restriction fragment length polymorphisms Difference between two samples of homologous DNA arising from differing locations of restriction sites

Crime scene Suspect w x y z Cut DNA from chromosomes C G A T

After digestion by restriction enzymes the fragments are run through a gel – + Longer fragments Shorter x w y z 1 2 Figure 12.11B

DNA profiling Crime scene Suspect 1 Suspect 2 DNA isolated DNA of selected markers amplified Amplified DNA compared 3

Used in forensic investigations STR site 1 STR site 2 Crime scene DNA Number of short tandem repeats match Number of short tandem repeats do not match Suspect’s DNA

Genomics Studying the entire genome and their interactions

Human Genome Project identify all the approximately 20,000-25,000 genes in human DNA, determine the sequences of the 3 billion chemical base pairs that make up human DNA, store this information in databases, improve tools for data analysis, transfer related technologies to the private sector, and address the ethical, legal, and social issues (ELSI) that may arise from the project.

Exons (regions of genes coding for protein Fig. 12-18 Exons (regions of genes coding for protein or giving rise to rRNA or tRNA) (1.5%) Repetitive DNA that includes transposable elements and related sequences (44%) Introns and regulatory sequences (24%) Unique noncoding DNA (15%) Repetitive DNA unrelated to transposable elements (15%)

HGP 3.2 billion nucleotide pairs RNA splicing Non-coding ~21000 genes How are we so complex? RNA splicing Non-coding Repetitive DNA Telomeres Transposable elements

Shotgun Method Chop up genome Clone fragments Sequence Squeeze it through a small, pressurized syringe Clone fragments Insert them into a vector Sequence Search for overlapping segments Reassemble the overlaps

Chromosome Chop up with restriction enzyme DNA fragments Sequence fragments Align fragments Reassemble full sequence

Recombo DNA http://www.youtube.com/watch?v=-sI5vy-cD2g&feature=related PCR http://www.youtube.com/watch?v=2KoLnIwoZKU&feature=related PCR and Gel http://www.youtube.com/watch?v=_uoPkavtXgs

Videos Ch Overview http://www.youtube.com/watch?v=Nc3jArZXHjs