FROM GENE TO DNA TECHNOLOGY

1. FROM GENE TO DNA TECHNOLOGY Case Study: Familial HYPERCHOLESTEROLEMIA RFLP gene mapping cDNA PCR in-situ hybridization gene cloning “knock-out” genes gene therapy References: The information about “FH” on these slides is excerpted from Online Mendelian Inheritance in Man http://www.ncbi.nlm.nih.gov/Omim/

2. Familial HYPERCHOLESTEROLEMIA Background an autosomal dominant disorder characterized by elevation of serum cholesterol bound to low-density lipoprotein (LDL), resulting in markedly increased cholesterol level and an increased incidence of early onset of atherosclerosis and its complications. Mutations in the LDL receptor (LDLR) gene on chromosome 19 cause this disorder. Occurance: about 7 out of 1000 people.

3. The LDL receptor was discovered through an extraordinary collaboration between two extraordinary scientists, Michael S. Brown and Joseph L. Goldstein.

4. Cells that need cholesterol synthesize LDL receptors. from Alberts et al. Molecular Biology of the Cell, Garland Publishing, Third edition, 1994.

5. Clathrin Pits

7. Mutations in the LDL receptor (LDLR) gene Horsthemke et al. (1987) suggested that unequal crossing-over between 2 Alu-repetitive DNA sequences was responsible for an intragenic deletion of the LDLR gene leading to familial hypercholesterolemia……..thus supporting a notion of recombination hotspots which involve Alu sequences. The deletion was presumably caused by an unequal crossover event between 2 homologous chromosomes at meiosis. A mutant LDL receptor with ARG 158 replaced by CYS.

8. A mutant LDL receptor with ARG 158 replaced by CYS. How was DNA technology used to: Locate the LDL receptor (LDLR) gene? Identify individuals who carry LDLR mutations? Study the disease using an animal model? Develop an ex vivo genetic therapy for this disease?

9. Gene Mapping The LDLRgenewas regionalized to 19p13.1-p13.3 by in situ hybridization (Lindgren et al., 1985). Judging by the sequence of loci suggested by linkage data (pter--FHC--C3--APOE/APOC2), the location of FHC (LDLR) is probably 19p13.2-p13.12 and of C3, 19p13.2-p13.11. Humphries et al. (1985) found a RFLPof the LDL receptor gene using the restriction enzyme PvuII.

10. Restriction Fragment Length Polymorphism (RFLP) analysis is a technique in which DNA samples may be differentiated by patterns derived from cleavage by a particular restriction endonuclease. RFLP If there are random polymorphisms that affect the pieces of DNA derived from restriction digestion, the length of the fragments produced will differ when the DNA is digested with a restriction enzyme.

11. RFLP Detection of RFLPs Most polymorphisms exist in non coding DNA. If a polymorphism affects a restriction “cut” site, then there will be two different sized DNA pieces that correspond to that site. 1) Cut the DNA sample with a particular restriction enzyme. 2) Run the cut pieces on an electrophoresis gel. 3) Stain the gel using a “probe” that is specific for the specific RFLP.

12. Gel Electrophoresis

13. Restriction Endonucleases Restriction endonucleases cut DNA at specific sites by hydrolysis of phosphodiester bonds Four to eight (usually six) nucleotide sequences are recognized by the nucleases The nucleases can cut straight across doubled stranded DNA to produce blunt ends or staggered.

14. Restriction Endonucleases

15. Interpretation of RFLPs RFLP 1) Shorter DNA pieces run toward the bottom of the gel. 2) Longer DNA pieces run toward the top. 3) The only visible pieces are those that bind to the probe.

16. MITOMAP: Polymorphic MtDNA Restriction Sites • Detected by High Resolution Screening of Human Populations • (Last edited Jun 07, 1999) • Enzyme Cut Sequence High Resolution Restriction Sites References • Acc I GTVWAC -15254 references • Alu I AGCT +259, +675, -856, -1240, +1403/1448, -1610, -1670, +1718, -1893, -1917, -2208, 5176, -5584, -5697, -5823, -5978, -5996, -6022, -6204, -6867, +7025, -7055, 9644, +10028, +10135, +10143, -10352, +10397, +10413/10536, -10598, +10694, +12990/12993/12996/13594, +13068, +13262, +13284, -14015, -14304, +14322, • Ava II GGRCC +748, +4332, +4481/10933, +5259, +5984, +6332, +6581, +6699/8719/8723, • 13367, +15487, +15591, +15882, -16390 references • BamHI GGATCC +13366, -14258, +16389 references • BstNI CCRGG +13467, -13704 references • Dde I CTNAG +29, +64, -853, +868, +1043, -1637, -1667, -1715, -1923, +2247, -2856, -3192, +3388, +9147, -9272, -9500, -9641, +10394, -10631, +10746, +11074, +11146, +11793, +13467, +15660, +15727, • EcoRI GAATTC No polymorphic sites detected references • FnuDII CGCG +14678/14680/14695 references • Hae II PGCGCQ +1622, -4529, +4830, -9052, +9326, +11001, +11968, +12949, -14858, Data from the “mtDNA database”

17. Notes: Employ numerous endonucleases. Separate the fragments on acrylamide gels. or Use PCRamplification, then separate on high resolution gels.

18. Diagnosis of Sickle-Cell Anemia (by RFLP) Individual with normal hemoglobin Individual with sickle cell hemoglobin Restriction cut site is mutated so that the resultant DNA fragment that binds to the probe is longer.

19. What would a heterozygous carrier look like? Individual with normal hemoglobin Individual with sickle cell hemoglobin

20. In situ Hybridization In general, nucleic acid hybridization is based on the principle that complementary sequences (DNA or RNA) will base pair with each other to form a stable molecule This principle is used in variety of biotechnology protocols including sequencing, PCR, and Southern blotting

22. Southern Blotting DNA is cut with restriction nucleases and separated by gel electrophoresis. DNA is denatured within the gel and transferred (blotted) to nitrocellulose or nylon membranes. Labeled DNA probes are allowed to hybridize to the blot. Labeled bands are visualized by autoradiography.

24. Diagnosis Li et al. (1988) worked out a PCR method for analyzing DNA sequences in individual diploid cells and human sperm. Recent progress in PCR technology and and mutation detection methodology have facilitated the rapid screening process for detecting these point mutations among a high number of patients.

25. PCRThe Polymerase Chain Reaction

27. PCR for Medical Diagnostics

28. PCR for Genetic AnalysisEthical Issues A number of newly discovered genes or mutations of genes are correlated with diseases such as cancer. PCR combined with DNA sequencing can can be used to identify individuals with these genes. Should this analysis be performed for a disease without a cure? Who should have access to this information?

30. Diagnosis of Sickle-Cell Anemia (by PCR)

31. PCR for Cloning

32. …….....The accumulation of cholesterol in patients suffering from FH was suspected to be caused by a defect in the protein LDL-receptor the main function of which was known to lie in the mopping up cholesterol from the circulating blood. In the next step the LDL-receptor gene was cloned from a unaffected individual using its affinity for cholesterol as screening procedure. The final proof for an implication of this protein in the pathogenesis in FH sufferers came from the demonstration that the LDL-receptor gene was mutated in FH-sufferers……………..

33. Cloning DNA

34. Mechanics of Cloning DNA How to identify the segment of DNA to be cloned? How to cut out (excise) the segment? What are the essential properties of a vector? How to insert a DNA segment into a vector? How do you know the segment of DNA has been inserted into the vector and in what orientation?

35. Mechanics of Cloning DNA What are the molecular tools required to clone a segment of DNA? Cloning can be defined as the removal (excision) of a segment of DNA (usually a gene) from one genome and insertion of that segment into a vector, thereby creating a recombinant DNA molecule that can replicate in a host cell.

37. Vector Properties Origin of replication: Must be able to replicate in a host cell Cloning Site: Must have a restriction nuclease site for cloning, usually a unique site (occurs only once in the vector) Selectable Marker: Must have a mechanism to select cells that contain the vector (usually antibiotic resistance)

38. DNA Ligase

39. DNA Restriction Mapping A physical map of a segment of DNA can be created by locating various restriction sites as landmarks Insertion of DNA into this segment will change the map relative to the site of insertion and can be used to “map” the insert

40. Constructing a “Knock-out Mouse DNA Construct Mouse LDL (low density lipoprotein) receptor cDNA was amplified by PCR from mouse liver first strand cDNA using polyA+ RNA and the following primers: Primer A (5'-ATTCT....

41. Low density lipoprotein receptor Normal fertility and normal triglyceride concentrations. Increased susceptibility to gross atheromata and to thickening of aortic valve leaflets. Increased susceptibility to increased plasma VLDL, IDL, and LDL cholesterol and total plasma cholesterol concentration. Increased susceptibility to develop massive xanthomatousinfiltration of skin and subcutaneous tissues

42. Wilson et al. (1992) presented a detailed clinical protocol for the ex vivo gene therapy of familial hypercholesterolemia. Their approach involves recovery of hepatocytes from the patient and reimplanting them after genetic correction by a retrovirus-mediated gene transfer. Not only were the technical details of vectors and viruses, transduction and delivery of hepatocytes, evaluation of engraftment and rejection, etc., discussed, but also assessment of risks versus benefits. Gene Therapy

43. Homework Recap Worksheet on DNA Technology PCR in situ hybridization cDNA cloning restriction mapping “Knock-out” mice RFLP polymorphisms restriction endonucleases gel electrophoresis

44. Dideoxy Sequencing of DNA The dideoxy or enzymatic method of DNA sequencing utilizes the principle that a dideoxyribonucleotide triphosphate can be incorporated into a growing DNA chain, but can not continue synthesis. DNA synthesis is terminated and the type of dideoxyNTP (ddNTP) added reflects the last nucleotide incorporated.

45. Dideoxy Sequencing of DNA

46. Reaction Requirements forDideoxy Sequencing of DNA Single-stranded DNA molecule (template) to be sequenced Oligonucleotide primer complementary to upstream region of template DNA polymerase All four dNTPs (dATP, dGTP, dCTP, dTTP) One of the four ddNTPs (ddATP, ddGTP, ddCTP, or ddTTP)