1 Gene Therapy Gene therapy: the attempt to cure an underlying genetic problem by insertion of a correct copy of a gene. –Tantalizingly simple and profound in theory, maddeningly difficult to actually achieve. –Easiest targets: access to or retrieval of cells Respiratory and blood Engineered virus, infects cells, carries in good gene. Engineer cell in tube, return to body.

2 Recent successes Adenosine Deaminase deficiency –Defect in nucleoside metabolism especially affects white blood cells X-linked chronic granulomatous disease –Neutrophils fail to make superoxide General scheme: Retrovirus used to replace gene in bone marrow cells, return cells to patient

3 Failures Genes don’t always make into genome –“cure” is short-lived as DNA disappears Viruses carrying genes insert in bad places –Cause over-expression of genes or DNA deletions Cancer Immune system becomes sensitized to vector Death of patient in clinical trial in 1999.

4 New development Sleeping Beauty transposon system –First transposon available in vertebrates? –Originally from inactive fish transposon Engineer with gene of interest –Transposon inserts with gene, getting it into the chromosome –Inserts in different places from viral vector –Much higher rate of gene insertion

5 Sleeping Beauty Transposon system Active transposon engineered from an inactive fish transposon. Transposon jumps into chromosome bringing good gene with it.

6 DNA Polymorphisms: DNA markers a useful tool in biotechnology Any section of DNA that varies among individuals in a population, “many forms”. Examples include: SNPs, RFLPs, STRPs, and AFLPs; –RFLPs include VNTRs and STRPs –microsatellites (STRs) = SSLPs = STRPs = SSRs Useful for finding, mapping genes involved in disease, and –Individual identification, epidemiology, anthropology, population/ecology studies, taxonomy.

7 SNPs Single nucleotide polymorphisms: regions of DNA where one base pair is different. Occur evenly spread over all the DNA. 1/ bp Detected by sequencing. If SNP occurs in a restriction enzyme site, it generates an RFLP. Could be in coding or non-coding regions. Over 300,000 human SNPs known and are being mapped.

8 SNP

9 RFLPs scidiv.bcc.ctc.edu/.../lectures/ DNATechnology/image021.jpg Restriction fragment length polymorphism. Mutation at a restriction site prevents recognition & cutting. Results in one band of larger DNA instead of 2 smaller ones.

10 Other RFLPs: VNTRs and STRPs Minisatellites and Microsatellites These are RFLPs because they are defined by or visible following restriction enzyme cuts. –Variable Number Tandem Repeats Groups (10-100) of nucleotides repeated 2 – 100 times (depending on individual and locus). Restriction sites on both sides of repeated DNA The more repeats, the longer the fragment. –Simple Tandem Repeat Polymorphisms Shorter, 2-9 nucleotides repeated Small enough number for PCR amplification Also called STRs, SSLPs, etc.

11 Use of VNTRs Restriction sites are on either side; fragment length depends on number of repeats in between sites.

12 STRPs Primers for both sides of repeated region allow PCR amplification of DNA; generates PCR products that differ in length depending on number of repeats. Becoming the standard method for DNA testing in forensics labs. Cheaper, easier, more sensitive.

13 STRs in forensics Locus vWA Alleles in different ethnic and racial groups examined, used as database. Panel of 13 different STRs are used. Because the odds of a particular combination of the 13 is product of the frequencies, numbers like 1 in 10 billion can be generated. Bandfrequency

14 THE 13 CODIS STRs STR African-AmericanU.S. Caucasian D3S vWA FGA TH TPOX CSF1PO D5S D13S D7S D8S D21S D18S D16S

15 RAPD: using PCR to find polymorphisms “Random amplified polymorphic DNA” Screen DNA from individuals by doing PCR with random short primers. By random chance, primers will amplify many different sections of DNA. Look for bands on gel that are not present in each individual tested. avery.rutgers.edu/.../ archives/onions/rapd.html

16 RAPD: using PCR to find polymorphisms-2