1. Recombinant DNA Technology Stephen B. Gruber, MD, PhD Division of Molecular Medicine and Genetics November 4, 2002

2. Learning Objectives Know the basics of gene structure, function and regulation. Be familiar with the basic methods of molecular genetics. Understand the meaning of DNA sequence and amino acid polymorphisms. Know how DNA sequence analysis is performed and be familiar with methods of screening for differences. Have a general understanding of methods for gene transfer into tissue culture cells and the power of transgenic technologies.

3. Learning Objectives (1) Know the basics of gene structure, function and regulation. Be familiar with the basic methods of molecular genetics. Understand the meaning of DNA sequence and amino acid polymorphisms. Know how DNA sequence analysis is performed and be familiar with methods of screening for differences. Have a general understanding of methods for gene transfer into tissue culture cells and the power of transgenic technologies.

4. Chromosomes, DNA, and Genes Cell Nucleus Chromosomes Gene Protein Adapted from Understanding Gene Testing, NIH, 1995

5. Genetic Code A codon is made of 3 base pairs 64 codons total 1 codon (AUG) encodes methionine and starts translation of all proteins 3 codons stop protein translation 61 codons encode 20 amino acids (redundant code) UA A A UG Met G C A Ala

6. DNA Transcription and Translation mRNA Ribosome Growing chain of amino acids Protein Nuclear membrane Cell membrane DNA Adapted from Understanding Gene Testing, NIH, 1995

7. 5' end Promoter RNA transcription start site 3' end Gene Structure Stop site IntronExon 2IntronExon 1Exon 3 Splice sites Exon 2Exon 1Exon 3 mRNA

8. RNA Processing Translation Protein DNA Primary mRNA Mature mRNA Processing Transcription ExonIntronExonIntronExon GU AG

9. Learning Objectives (2) Know the basics of gene structure, function and regulation. Be familiar with the basic methods of molecular genetics. –nucleic acid hybridization –Southern (DNA) and northern (RNA) blotting –PCR –DNA sequencing –basic steps involved in constructing & screening a cDNA library Understand the meaning of DNA sequence and amino acid polymorphisms. DNA sequence analysis Transgenic technologies

10. from Textbook: 5.4 1944 DNA is the genetic material 1949 Abnl Hemoglobin in sickle cell anemia 1953 Double helix 1956 Glu 6 Val in sickle hemoglobin 1966 Completion of the genetic code 1970 First restriction enzyme 1972 Recombinant plasmids 1975 Southern blotting 1981 Transgenic mice 1983 Huntington Disease gene mapped 1985 PCR 1986 Positional cloning (CGD, muscular dystrophy, retinoblastoma 1945 19501955 1960 1965 1970 19751980 19851990 1995 2000 1987 Knockout mice 1989 Positional cloning without deletion (CF) 1990 First NIH- approved gene therapy experiment 1996 Complete yeast genome sequence 1995 1 st complete bacterial genome sequence 2001 Draft human genome sequence

11. Preparing DNA for Analysis Blood sample Centrifuge and extract DNA from white blood cells DNA for analysis

12. Textbook: Figure 5.8

13. Electrophoresis of DNA Voltage + DNA fragments loaded into wells Path of migration DNA fragments separate by size and charge _

14. Electrophoresis Restriction enzyme digestion Principle of a Southern blot hybridize labeled probe to fragment of DNA Add radio-labeled normal DNA probes

15. Polymerase Chain Reaction (PCR) Isolate and denature DNA Anneal and extend primers Repeat as necessary Amplified segments Sequence to be amplified

16. DNA Sequencing

17. Textbook: Figure 5.17

18. DNA Sequencing ATC TTA GAG TGT CCC ATC TTA GTG TCC C Start A T C G Normal Mutant (185delAG) AG A T C G delA Start delG

19. Learning Objectives (3) Know the basics of gene structure, function and regulation. Be familiar with the basic methods of molecular genetics. –nucleic acid hybridization –Southern (DNA) and northern (RNA) blotting –PCR and gel electrophoresis –DNA sequencing –basic steps involved in constructing & screening a cDNA library Understand the meaning of DNA sequence and amino acid polymorphisms. DNA sequence analysis Transgenic technologies

20. Polymorphisms and Mutations Sequence variation-- differences among individuals (DNA, amino acid) –> 0.01 = polymorphism –< 0.01 = rare variant Mutation-- any change in DNA sequence –Silent vs. amino acid substitution vs. other –neutral vs. disease-causing Common but incorrect usage: “mutation vs. polymorphism” balanced polymorphism= disease + polymorphism

21. Learning Objectives (3) (continued) Understand the meaning and significance of DNA sequence and amino acid polymorphisms. Understand the various types of DNA sequence polymorphisms. –RFLPs (Restriction Fragment Length Polymorphism) –VNTRs (Variable Number Tandem Repeat) –SSRs (Simple Sequence Repeat; also STR [Short/Simple Tandem Repeat])) –SNPs (Single Nucleotide Polymorphism)

22. Textbook: Figure 5.19

23. Learning Objectives (3) (continued) Understand the meaning and significance of DNA sequence and amino acid polymorphisms. Understand the various types of DNA sequence polymorphisms. –RFLPs (Restriction Fragment Length Polymorphism) –VNTRs (Variable Number Tandem Repeat) –SSRs (Simple Sequence Repeat; also STR [Short/Simple Tandem Repeat])) –SNPs (Single Nucleotide Polymorphism)

24. Disease-Associated Mutations Alter Protein Function Functional protein Nonfunctional or missing protein

25. P1P2 (TCTA) 10 (TCTA) 11 (TCTA) 12 (TCTA) 13 (TCTA) 14 (TCTA) 15 A B C D E F ABCDEFAFCE 15 14 13 12 11 10 Textbook: Figure 5.22

26. SNP (coding sequence) Normal mRNAProtein A UG Met A A G Lys UU U Phe G G C Gly G C A Ala U U G Leu A A Gln C Silent DNA sequence polymorphism Sequence variant mRNAProtein A UG Met A A G Lys UU U Phe G G U Gly G C A Ala U U G Leu A A Gln C G

27. Disease-Associated Mutations A mutation is a change in the normal base pair sequence Commonly used to define DNA sequence changes that alter protein function

28. Polymorphism DNA sequence changes that do not alter protein function (common definition, not technically correct) Functional protein

29. Polymorphism Variation in population –phenotype –genotype (DNA sequence polymorphism) Variant allele > 1% “Normal” Disease < 1%> 1% Rare or “private” polymorphism Common usage: disease ?? Factor V R506Q: thrombosis, 3% allele frequency

30. THE BIG RED DOG RAN OUT. THE BIG RAD DOG RAN OUT. THE BIG RED. THE BRE DDO GRA. THE BIG RED ZDO GRA. Mutations Normal Missense Nonsense Frameshift (deletion) Frameshift(insertion) Point mutation: a change in a single base pair

31. Silent Sequence Variants Normal mRNAProtein A UG Met A A G Lys UU U Phe G G C Gly G C A Ala U U G Leu A A Gln C Sequence variant: a base pair change that does not change the amino acid sequence (a type of polymorphism) Sequence variant mRNAProtein Adapted from Campbell NA (ed). Biology, 2nd ed, 1990 A UG Met A A G Lys UU U Phe G G U Gly G C A Ala U U G Leu A A Gln C G

32. Missense Mutations Missense Missense: changes to a codon for another amino acid (can be harmful mutation or neutral polymorphism) mRNAProtein Normal mRNAProtein A UG Met A A G Lys UU U Phe G G C Gly G C A Ala U U G Leu A UG Met A A G Lys UU U Phe A G C Ser G C A Ala U U G Leu A A Gln CA A C Adapted from Campbell NA (ed). Biology, 2nd ed, 1990

33. Nonsense Mutations Nonsense: change from an amino acid codon to a stop codon, producing a shortened protein Nonsense mRNAProtein Normal mRNAProtein A UG Met A A G Lys UU U Phe G G C Gly G C A Ala U U G Leu A UG Met U A GUU U G G C G C AU U G A A Gln CA A C Adapted from Campbell NA (ed). Biology, 2nd ed, 1990

34. Frameshift Mutations Frameshift U GC A A A UG Met A A G Lys G C G Ala C AU UU U G Leu Frameshift: insertion or deletion of base pairs, producing a stop codon downstream and (usually) shortened protein mRNAProtein Normal mRNAProtein A UG Met A A G Lys UU U Phe G G C Gly G C A Ala U U G Leu A A Gln C Adapted from Campbell NA (ed). Biology, 2nd ed, 1990

35. Splice-Site Mutations Exon 1 Intron Exon 2 Intron Exon 3 Exon 1 Exon 3 Altered mRNA Splice-site mutation: a change that results in altered RNA sequence Exon 2

36. Other Types of Mutations Mutations in regulatory regions of the gene Mutations in regulatory regions of the gene Large deletions or insertions Large deletions or insertions Chromosomal translocations or inversions Chromosomal translocations or inversions

37. Types of Mutations Point Mutations –Silent –Missense –Nonsense –(frameshift) Deletion/Insertion –small –large Rearrangement Transcription RNA Processing –splicing –poly A –RNA stability Protein level –processing –stability –altered function gain loss new

38. Learning Objectives (4) Know the basics of gene structure, function and regulation. Be familiar with the basic methods of molecular genetics. Understand the meaning of DNA sequence and amino acid polymorphisms. Know how DNA sequence analysis is performed and be familiar with methods of screening for differences. –SSCP –DGGE –CSGE –ASO –Chip technology methods for gene transfer and the power of transgenics

39. Tests to Detect Unknown Mutations Used when a specific mutation has not been previously identified in a family DNA sequencing is most informative method Simpler scanning tests also may be used, usually followed by limited sequencing to characterize the specific mutation

40. Single Strand Conformational Polymorphism (SSCP) DNA Gel NormalMutated mutation DNA is denatured into single strands Single strands fold; shape is altered by mutations Mobility of mutant and normal strands differ in gel

41. Evaluating SSCP Pros Rapid, simple, and widely available for many genes Detects 60%  95% of mutations in short DNA strands Cons Subsequent DNA sequencing needed to characterize mutation Sensitivity drops with longer DNA sequences

42. Denaturing Gradient Gel Electrophoresis (DGGE) DNA denatured into single strands Single strands reanneal into normal and mutant homoduplexes and heteroduplexes Hetero- and homoduplexes denature at different points in gradient gel DNA Denaturing gradient gel NormalMutated

43. Denaturing Gradient Gel 1 normal homoduplex band 2 heteroduplex bands 1 mutant homoduplex band BRCA1 mutation carrier

44. Evaluating DGGE Pros Highly sensitive (>90%) Better resolution than SSCP Cons Not efficient for analyzing large DNA fragments Subsequent DNA sequencing needed to characterize mutation Labor-intensive set-up

45. Heteroduplex Analysis (CSGE) Normal band Mutated bands Single-strand DNA Cold Reannealed DNA Amplify and denature DNA

46. Evaluating Heteroduplex Analysis Pros >90% sensitivity Rapid, simple assay Easily automated for high throughput use Cons Subsequent sequencing needed to characterize mutation

47. Tests to Search for Known Mutations Used when a specific mutation is known or suspected to occur in a family Methods focus on detection of one or a few specific mutations (eg, “Ashkenazi Jewish panel”) Methods include ASO, CSGE, restriction site digestion, others

48. Add radio-labeled normal DNA probes Amplify DNA and hybridize to membranes Allele Specific Oligonucleotide (ASO) Hybridization Add known mutant DNA probes Patients #1#2#3 #1#2#3

49. Evaluating ASO Analysis Pros Sensitive method to detect known mutations Panels of ASO probes useful to detect common mutations Cons Each ASO probe detects only one specific sequence Most useful for small sequence changes

50. Principle of Microarray (Chip) Assay Synthetic DNA probes PrehybridizationPosthybridization Probes with hybridized DNA

51. Mutation vs. Silent Sequence Variation Obvious disruption of gene –large deletion or rearrangement –frameshift –nonsense mutation Functional analysis of gene product –expression of recombinant protein –transgenic mice New mutation by phenotype and genotype X

52. Learning Objectives (5) Know the basics of gene structure, function and regulation. Be familiar with the basic methods of molecular genetics. Understand the meaning of DNA sequence and amino acid polymorphisms. Know how DNA sequence analysis is performed and be familiar with methods of screening for differences. Have a general understanding of methods for gene transfer into tissue culture cells and the power of transgenic technologies.

54. Summary Gene structure helps us understand where to look for errors. PCR and gel electrophoresis essential for diagnostic tests. DNA polymorphisms are best defined by frequency. Screening for DNA sequence differences is performed by direct sequencing or other techniques that are selected based on whether the mutation is known or unknown. Introduction to gene transfer provides a framework for learning about gene therapy and methods for recombinant drug development.