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Chapter 15 Biotechnology.docx

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Temple University
BIOL 1001

Final Exam Study Guide Chapter 15- Biotechnology Classic biotechnology – fermentation Recombinant DNA– splicing genes from one organism into another (produces a transgenic organism not normally found in nature). Enzymes Needed: Rest Enzymes, DNAligase Restriction Enzymes • Cut double-stranded DNAinto fragments (fragments base-pair at sticky ends), some restriction enzymes give rise to blunt ends too • Do not end in the suffix –ase. • Recognize palindromic sequences in the DNA • DNALigase Enzyme seals up breaks in both strands of the DNA Plasmids • Cloning Vectors • Small circle of bacterial DNAoutside the chromosome, give rise to bacterial resistance • Foreign DNAcan be inserted into a plasmid –Forms recombinant plasmids • Can be used to deliver DNAinto another cell, or cell of another organism. • Cut plasmid and foreign DNAwith same restriction enzyme Polymerase Chain Reaction • Sequence to be copied is heated to denature DNA • Single-stranded DNAprimers added, and bind to ends of both single strands of template DNA • DNApolymerase uses free nucleotides to create complementary strands • Taq DNAPolymerase is used (heat stable) • Doubles number of copies of DNAevery cycle DNAsequencing • Once you know the DNAsequence, can develop a genetic test (Huntington’s disease, PKU) • Nucleotides are assembled to create complimentary strands. • When a modified nucleotide is included, DNAsynthesis stops (dideoxy method) • Result is millions of tagged copies of varying length Human Genome Project (HUGO) • Map entire human genome by dideoxy sequencing • James Watson was first director • Process accelerated when Craig Ventner used bits of cDNAs as hooks to find genes (shotgun technique). • Coding regions (exons of genes) make up only 1.5% of our DNA. • Transposons make up 42% of our DNA • About half of remaining DNAis repeated segments that are derived from transposons. • Around 1.4 million SNPs in the human genome (single nucleotide polymorphisms that vary by one base change). • SNP: The bitter taste receptor for PTC shows polymorphisms at three different sites in the coding region of the gene Next Gen DNASequencing • Allows rapid and repeated sequencing of human DNAby parallelizing the sequencing process • Produce thousands or millions of DNAsequences at once • Used to predict certain diseases, preventative medicine • Correlate specific genes with medical histories • Challenge is to deliver the value of DNAsequence data out to physicians DNAFingerprints • Unique array of DNAfragments • Inherited from parents in medelian fashion • All individuals have a unique DNAfingerprint except for identical twins • Uses highly repeated microsatellite DNA • USES: paternity suits, capital crimes (murder & rape), identify relatives, classification of organisms DNAFingerprint via STR • 3 million bases differs in every person • RFLP testing: requires much more DNA, DNAcan’t be degraded, rarely used anymore • PCR-Based testing: need little DNA, prone to contamination especially at crime scene. • PCR is used to amplify specific regions of DNAwith known variations • 12 million monozygotic twins on the earth today Short Tandem Repeats in DNA • Most of our DNAis the same, 99.9% • Short regions of DNAthat differ substantially among individuals (2-5 nt long) • Adjacent to eachother • Occur in many sites on genome • Each person carries a unique combination of repeat numbers • Tetra- or Penta-nucleotide repeats used for DNAfingerprinting • 13 different tandem repeats used in crime labs, plus amelogenin VNTR • Alocation in a genome where an STR is organized as a tandem repeat (in a region that does not encode genes) • Can be found on many chromosomes, variations in length between individuals • Often found in INTRONS of genes • Use PCR to amplify these tandem repeats using primers • Each VNTR variant acts as an inherited allele from mom & dad • Microsatellites are 1 to 6 nucleotide repeats that are dispersed throughout our chromosomes STR • Type of VNTR • 3-6 bases long • Found in microsatellite data • In agarose gel, each subject has 2 bands, one from mom, one from dad • Can get either one band or two bands at a locus CODIS • FBI databank is know as Combined DNAIndex System (CODIS), holds DNAprofiles • CODIS uses 13-15 sets of STRs for DNAfingerprinting • CODIS identifies 15 marker plusAMEL to determine sex AMEL-Amelogenin Gene • Encodes amelogenin protein, used to produce tooth enamel • Gene has a locus on both the X and Y chromosome • And use INTRON 1 of amelogenin gene for sex determination (allele on Y chromosome has 112 base pair size for intron 1) • NativeAmericans have deletions in amelogenin male gene DNAFingerprinting RFLPS • Need a larger amount of DNA • DNAfrom areas with tandem repeats is amplified by PCR, then cut with restriction enzymes • Resctriction Fragments vary in size • Gel electrophoresis Mitochrondrial DNAFingerprint • Mitochondria have their own DNA, circular genome with 16,500 bases • Mitochondrial DNA is maternally inherited, mother and all her children have the same DNA • Used with bones, calcified tissues • Use RFLP analysis (hypervariable regions 1 and 2 (HRV) • Many copies of Mitochondrial DNAper cell, and per mitochondria ChromosomeAnalysis used for Geneology • Useful in researching direct paternal line, 86 genes on Y • The Y chromosome has a lot of non-coding DNA, short tandem repeats • Analysis of genetic markers on the Y chromosome is especially useful for tracing relationships among males.Analyzing biological evidence involving multiple male contributors • DYS loci consist of short segments of repeated DNA(STR) • SNP testing of the Y-chromosome Innocence Project • First Conviction overturned by DNAevidence in 1993 Human Gene Therapy • Viruses are usually used to insert genes into cultured human cells • Vectors for eukaryotic genes include: weakened adenovirus, weakened adeno-associated virus, weakened lentiviruses, naked DNAcoated with lipids • When integrated into host chromosome very difficult to get modified genes to work where they should • Used to treat cancers and autosomal recessive disorders like cystic fibrosis, control retinal disease (blindness) • ADENOVIRUS is vector for human gene therapy, weakened so it can’t cause disease • LIPOPLEXES for delivering DNA, coat DNAwith lipids so its protected from degradation Genes of Death Reading • Gene therapy took off in 1990, geneticist William FrenchAnderson reported he cured a girl of SCID • Pennslyvania’s Institute for Human Gene Therapy, led by James Wilson, led team to development OTC cure (OTC: livers cannot metabolize ammonia that is produced when you digest protein, goes into blood, causes coma and death) • Engineered an adenovirus to carry normal version of defective gene, hope that liver cells would use it • OTC deficient adults are mostly women (X-linked disorder) • Jesse Gelsinger, boy living with it, killed after treatment • Vector killed 3 monkeys so Wilson said he made a safer one, but there was no proof • Worked on mice and women
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