Recombinant DNA Technology

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

2 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. hromosomes, DNA, and Genes Gene ell Nucleus hromosomes Protein Adapted from Understanding Gene Testing, NIH,

3 Genetic ode A codon is made of 3 base pairs 64 codons total 1 codon (AUG) encodes methionine and starts translation of all proteins 61 codons encode 20 amino acids (redundant code) 3 codons stop protein translation A U G G A U A A Met Ala DNA Transcription and Translation Growing chain of amino acids Ribosome Protein DNA Nuclear membrane Adapted from Understanding Gene Testing, NIH, 1995 ell membrane 3

4 Gene Structure RNA transcription start site Splice sites Stop site Promoter Exon 1 Intron Exon 2 Intron Exon 3 5' end 3' end Exon 1 Exon 2 Exon 3 RNA Processing DNA Primary Exon Intron Exon Intron Exon Mature GU AG Transcription Processing Translation Protein 4

5 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 PR 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 1944 DNA is the genetic material 1953 Double helix 1956 Glu 6 Val in sickle hemoglobin 1970 First restriction enzyme 1983 Huntington Disease gene mapped 1981 Transgenic mice 1975 Southern blotting 1985 PR 1989 Positional cloning without deletion (F) 2001 Draft human genome sequence st complete bacterial genome sequence Abnl Hemoglobin in sickle cell anemia 1966 ompletion of the genetic code 1972 Recombinant plasmids 1986 Positional cloning (GD, muscular dystrophy, retinoblastoma First NIHapproved genome sequence omplete yeast gene therapy experiment 1987 Knockout mice from Textbook: 5.4 5

6 Preparing DNA for Analysis Blood sample entrifuge and extract DNA from white blood cells DNA for analysis SINGLE-STRANDED DNA PROBES FOR GENE A D + B MIXTURE OF SINGLE-STRANDED DNA MOLEULES E A F D B A B D A E E F F ONLY A FORMS A STABLE DOUBLE-STRANDED OMPLEXES A,, E ALL FORM STABLE OMPLEXES STRINGENT HYBRIDIZATION REDUED-STRINGENY HYBRIDIZATION Textbook: Figure 5.8 6

7 Electrophoresis of DNA DNA fragments loaded into wells _ DNA fragments separate by size and charge Path of migration Voltage + Principle of a Southern blot hybridize labeled probe to fragment of DNA Electrophoresis Restriction enzyme digestion Add radio-labeled normal DNA probes 7

8 Isolate and denature DNA Polymerase hain Reaction (PR) Anneal and extend primers Repeat as necessary Amplified segments Sequence to be amplified DNA Sequencing 8

9 5' SINGLE-STRANDED DNA OF UNKNOWN SEQUENE T G A T T G A A A 3' RADIOATIVELY LABELED T G T T PRIMER 3' 5' DNA POLYMERASE I datp dgtp dtp dttp P P P O H 2 O DIDEOXYNULEOTIDE (ddntp) H H H H H BASE H ddatp ddtp ddttp ddgtp REATION MIXTURES ddatp GEL ELETROPHORESIS AUTORADIOGRAPHY TO DETET RADIOATIVE BANDS ddtp ddttp ddgtp T G A T T G A A A ddg ddg ddg PRODUTS IN ddgtp REATION LARGER FRAGMENTS SMALLER FRAGMENTS T G A T T G READ SEQUENE OF ORIGINAL SINGLE-STRANDED DNA (OMPLEMENT OF PRIMER- GENERATED SEQUENE LADDER) Textbook: Figure 5.17 DNA Sequencing AG AT TTA GAG TGT AT TTA GTG T A T G A T G dela delg Start Normal Start Mutant (185delAG) 9

10 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 PR 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 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 ommon but incorrect usage: mutation vs. polymorphism balanced polymorphism= disease + polymorphism 10

11 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) Textbook: Figure

12 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) Disease-Associated Mutations Alter Protein Function Functional protein Nonfunctional or missing protein 12

13 P1 (TTA) 10 (TTA) 11 (TTA) 12 (TTA) 13 (TTA) 14 (TTA) 15 P2 A B D E F AB D EF AF E Textbook: Figure 5.22 SNP (coding sequence) Normal Protein A U G Met A A G Lys U U U Phe G G Gly G A Ala U U G Leu A A Gln Sequence variant Protein A U G Met A A G Lys U U U Phe G G U Gly G A Ala U U G Leu A A Gln Silent DNA sequence polymorphism 13

14 Disease-Associated Mutations A mutation is a change in the normal base pair sequence ommonly used to define DNA sequence changes that alter protein function Polymorphism DNA sequence changes that do not alter protein function (common definition, not technically correct) Functional protein Functional protein 14

15 Polymorphism Variation in population phenotype genotype (DNA sequence polymorphism) Variant allele > 1% ommon usage: < 1% > 1% Normal Rare or private polymorphism polymorphism Disease disease?? Factor V R506Q: thrombosis, 3% allele frequency Mutations Normal Missense Nonsense Frameshift (deletion) Frameshift (insertion) THE BIG RED DOG RAN OUT. THE BIG RAD DOG RAN OUT. THE BIG RED. THE BRE DDO GRA. THE BIG RED ZDO GRA. Point mutation: a change in a single base pair 15

16 Silent Sequence Variants Normal Protein A U G Met A A G Lys U U U Phe G G Gly G A Ala U U G Leu A A Gln Sequence variant Protein A U G Met A A G Lys U U U Phe G G U Gly G A Ala U U G Leu A A Gln Sequence variant: a base pair change that does not change the amino acid sequence (a type of polymorphism) Adapted from ampbell NA (ed). Biology, 2nd ed, 1990 Missense Mutations Normal Protein A U G Met A A G Lys U U U Phe G G Gly G A Ala U U G Leu A A Gln Missense Protein A U G Met A A G Lys U U U Phe A G Ser G A Ala U U G Leu A A Gln Missense: changes to a codon for another amino acid (can be harmful mutation or neutral polymorphism) Adapted from ampbell NA (ed). Biology, 2nd ed,

17 Nonsense Mutations Normal Protein A U G Met A A G Lys U U U Phe G G Gly G A Ala U U G Leu A A Gln Nonsense Protein A U G Met U A G U U U G G G A U U G A A Nonsense: change from an amino acid codon to a stop codon, producing a shortened protein Adapted from ampbell NA (ed). Biology, 2nd ed, 1990 Frameshift Mutations Normal Protein A U G Met A A G Lys U U U Phe G G Gly G A Ala U U G Leu A A Gln U Frameshift A U G A A G U U G G G A U U G A A Protein Met Lys Leu Ala Frameshift: insertion or deletion of base pairs, producing a stop codon downstream and (usually) shortened protein Adapted from ampbell NA (ed). Biology, 2nd ed,

18 Splice-Site Mutations Exon 1 Intron Exon 2 Intron Exon 3 Exon 2 Altered Exon 1 Exon 3 Splice-site mutation: a change that results in altered RNA sequence Other Types of Mutations Mutations in regulatory regions of the gene Large deletions or insertions hromosomal translocations or inversions 18

19 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 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. SSP DGGE SGE ASO hip technology methods for gene transfer and the power of transgenics 19

20 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 DNA Single Strand onformational Polymorphism (SSP) Normal Mutated DNA is denatured into single strands Single strands fold; shape is altered by mutations Mobility of mutant and normal strands differ in gel Gel mutation 20

21 Evaluating SSP Pros Rapid, simple, and widely available for many genes Detects 60% 95% of mutations in short DNA strands ons Subsequent DNA sequencing needed to characterize mutation Sensitivity drops with longer DNA sequences DNA Normal Denaturing Gradient Gel Electrophoresis (DGGE) Mutated 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 Denaturing gradient gel 21

22 Denaturing Gradient Gel BRA1 mutation carrier 1 normal homoduplex band 2 heteroduplex bands 1 mutant homoduplex band Evaluating DGGE Pros Highly sensitive (>90%) Better resolution than SSP ons Not efficient for analyzing large DNA fragments Subsequent DNA sequencing needed to characterize mutation Labor-intensive set-up 22

23 Heteroduplex Analysis (SGE) Amplify and denature DNA Single-strand DNA old Reannealed DNA Mutated bands Normal band Pros Evaluating Heteroduplex Analysis >90% sensitivity Rapid, simple assay Easily automated for high throughput use ons Subsequent sequencing needed to characterize mutation 23

24 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, SGE, restriction site digestion, others Allele Specific Oligonucleotide (ASO) Hybridization Amplify DNA and hybridize to membranes Add radio-labeled normal DNA probes Add known mutant DNA probes Patients #1 #2 #3 #1 #2 #3 24

25 Evaluating ASO Analysis Pros Sensitive method to detect known mutations Panels of ASO probes useful to detect common mutations ons Each ASO probe detects only one specific sequence Most useful for small sequence changes Principle of Microarray (hip) Assay Prehybridization Posthybridization Synthetic DNA probes Probes with hybridized DNA 25

26 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 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. 26

27 ULTURED ES ELLS WITH TARGETED GENE ALTERATION REMOVE FERTILIZED OOYTES FROM OVULATING MOUSE IMMEDIATELY AFTER FERTILIZATION HOLDING PIPETTE FEMALE PRONULEUS REMOVE BLASTOYSTS FROM PREGNANT MOUSE FOUR DAYS AFTER OVULATION INJETION NEEDLE OOYTE IMPALING MALE PRONULEUS OF OOYTE AND INJETING DNA REIMPLANT SEVERAL OOYTES IN FOSTER MOTHER INJET ES ELLS INTO BLASTOYST REIMPLANT SEVERAL BLASTOYSTS IN FOSTER MOTHER BIRTH BIRTH A B D SOUTHERN BLOT OF TAIL DNA A B D NORTHERN BLOT A B D + BIRTH BREEDING A B D SOUTHERN BLOT OF TAIL DNA A B D NORMAL GENE ALTERED GENE Summary Gene structure helps us understand where to look for errors. PR 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. 27

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