1Gene 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.

2Recent 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

3Failures

• 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.

4New 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

5Sleeping Beauty Transposon system

Active transposon engineered from an inactive fish transposon.

Transposon jumps into chromosome bringing good gene with it.

http://www.discoverygenomics.net/sbts.html

6DNA 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.

7SNPs

Single nucleotide polymorphisms: regions of DNA where one base pair is different.

Occur evenly spread over all the DNA. 1/ 1000-3000 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

9RFLPs

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.

10Other RFLPs: VNTRs and STRPsMinisatellites 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.

11Use of VNTRs

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

12STRPs

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.

13STRs in forensics

Locus vWA

• 14 0.081

• 15 0.107

• 15.2 0.179

• 16 0.306

• 17 0.192

• 18 0.089

• 19 0.047

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.

Band frequency

14THE 13 CODIS STRs

STRAfrican-American U.S. Caucasian

D3S1358 0.102 0.078

vWA 0.058 0.065

FGA 0.035 0.036

TH01 0.102 0.094

TPOX 0.081 0.211

CSF1PO 0.070 0.122

D5S818 0.097 0.140

D13S317 0.131 0.074

D7S820 0.081 0.061

D8S1179 0.075 0.067

D21S11 0.033 0.045

D18S51 0.028 0.030

D16S539 0.066 0.103

http://expertpages.com/news/dna.htm

15RAPD: 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

16RAPD: using PCR to find polymorphisms-2