Recombinant Dna Technology Power Point by xrm18253

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									      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
                          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.




         Chromosomes, DNA, and Genes
                                                     Gene
             Nucleus
     Cell                Chromosomes




                                                            Protein

Adapted from Understanding Gene Testing, NIH, 1995




                                                                      2
                               Genetic Code
                          A codon is made of 3 base pairs
                                 64 codons total

  1 codon (AUG) encodes                 61 codons encode 20          3 codons stop
   methionine and starts                     amino acids                 protein
 translation of all proteins              (redundant code)             translation

            A U G                               G C A                    U A A

              Met                                    Ala




                    DNA Transcription and
                        Translation
                                                            Growing
                                                            chain of
                                                           amino acids



                      mRNA
                                                      Ribosome            Protein

                                Nuclear
                               membrane                             Cell membrane
    DNA
Adapted from Understanding Gene Testing, NIH, 1995




                                                                                     3
                       Gene Structure
         RNA transcription        Splice sites                   Stop site
            start site

Promoter
             Exon 1    Intron      Exon 2        Intron    Exon 3

5' end                                                                       3' end


                       Exon 1      Exon 2        Exon 3

                                   mRNA




                      RNA Processing
            Exon        Intron         Exon           Intron        Exon
 DNA
                                                 Transcription
 Primary
 mRNA
                             GU   AG
                                                 Processing
            Mature
            mRNA
                                                 Translation


                       Protein




                                                                                      4
                                           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




                                                                                            1983                              2001
                                                                                         Huntington                        Draft human
                                                                                        Disease gene                     genome sequence
                                                                                           mapped          1989
                                     1956                                             1981                 Positional cloning
                                     Glu 6 Val in                          Transgenic mice                 without deletion (CF)
                                     sickle hemoglobin
     1944                                                        1970           1975                                1995
                                                                                                   1985             1st complete
  DNA is the              1953                             First restriction    Southern           PCR
genetic material          Double helix                         enzyme           blotting                            bacterial
                                                                                                                    genome sequence


      1945         1950       1955        1960      1965         1970          1975     1980      1985     1990      1995          2000

                                                                 1972                              1986     1990         1996
                 1949                             1966                                                                   Complete yeast
                                                              Recombinant             Positional cloning    First NIH-
   Abnl Hemoglobin                     Completion of the                                                                 genome sequence
                                                               plasmids                (CGD, muscular       approved
in sickle cell anemia                     genetic code
                                                                                             dystrophy,     gene therapy
                                                                                        retinoblastoma      experiment

                                                                                                      1987
                                                                                                    Knockout
                                                                                                      mice



                                                                                                            from Textbook: 5.4




                                                                                                                                           5
        Preparing DNA for Analysis




Blood sample                 Centrifuge and                                  DNA for analysis
                           extract DNA from
                            white blood cells




               SINGLE-STRANDED
                 DNA PROBES
                 FOR GENE A
                                                   C

                                                                   D



                                               B                   MIXTURE OF
                                           +                       SINGLE-STRANDED
                                                                   DNA MOLECULES

                                                   E                   A



                                                           F




                                                               C
                   C



                               D

               B                                          B            D
                                       A                                             A

                       E
                                                               E

                                   F                                            F




                 ONLY A FORMS A STABLE                          A, C, E ALL FORM
               DOUBLE-STRANDED COMPLEXES                       STABLE COMPLEXES

                STRINGENT HYBRIDIZATION                REDUCED-STRINGENCY HYBRIDIZATION

                                                                                          Textbook: Figure 5.8




                                                                                                                 6
             Electrophoresis of DNA
                     DNA fragments loaded into wells
                                                          _
DNA fragments
separate by size
  and charge
                                                       Voltage
                              Path of migration


                                                         +




             Principle of a Southern blot
        hybridize labeled probe to fragment of DNA

                            Electrophoresis




Restriction enzyme
     digestion




                             Add radio-labeled
                               normal DNA
                                  probes




                                                                 7
        Polymerase Chain Reaction
                 (PCR)
  Isolate and      Anneal and     Repeat as   Amplified
denature DNA     extend primers   necessary   segments



Sequence to be
  amplified




                 DNA Sequencing




                                                          8
                                                                                         SINGLE-STRANDED DNA
                                                                                        OF UNKNOWN SEQUENCE
                                                                      5'
                                                                                   C T G A C T T C G A C A A
                                                                                                                                    3'


                                                                            RADIOACTIVELY LABELED
                                                                                   PRIMER         3' T G T T
                                                                                                                                   5'




                                                                  DNA POLYMERASE I             P   P   P     O   CH 2 O BASE
                                                                  dATP                                                     H
                                                                                                                       H
                                                                  dGTP                                             H           H
                                                                  dCTP                                                 H   H
                                                                  dTTP                       DIDEOXYNUCLEOTIDE (ddNTP)

                                                              ddATP        ddCTP     ddTTP    ddGTP




                                                                                                         REACTION
                                                                                                         MIXTURES




                                                                                             C T G A C T T C G A C A A
                                       GEL
                                 ELECTROPHORESIS
                                                                                                             ddG
                               AUTORADIOGRAPHY
                              ddATP

                                   TO DETECT                                                       ddG
                               RADIOACTIVE BANDS




                                                      ddGTP
                                      ddCTP


                                              ddTTP
                                                                                     ddG

                                                                                               PRODUCTS IN ddGTP REACTION


                    LARGER                                    C
                  FRAGMENTS                                   T
                                                              G
                                                              A            READ SEQUENCE OF ORIGINAL
                                                                           SINGLE-STRANDED DNA
                                                              C
                                                                           (COMPLEMENT OF PRIMER-
                                                              T            GENERATED SEQUENCE LADDER)
                                                              T
                                                              C
                   SMALLER                                    G
                  FRAGMENTS




                                                                                                                                         Textbook: Figure 5.17




                      DNA Sequencing
                                                                                                                                   AG
        ATC TTA GAG TGT CCC                                                         ATC TTA GTG TCC C

         A    T       C               G                                              A                   T                     C             G




                                                                                                                                                    delG
                                                      delA




Start         Normal                                              Start             Mutant (185delAG)




                                                                                                                                                                 9
                    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




         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




                                                                         10
            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 5.19




                                                                         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)




Disease-Associated Mutations
      Alter Protein Function




 Functional protein                        Nonfunctional or
                                           missing protein




                                                              12
                  P1                                        P2
                                    (TCTA)10                     A
                                    (TCTA)11                     B
                                    (TCTA)12                     C
                                    (TCTA)13                     D

                                    (TCTA)14                     E

                                    (TCTA)15                     F




                       15
                       14
                       13
                       12
                       11
                       10




                             AB     CD     EF   AF     CE
                                                                       Textbook: Figure 5.22




               SNP (coding sequence)


            mRNA
Normal                      A U G    A A G      U U U       G G C    G C A   U U G   C A A
            Protein          Met         Lys     Phe         Gly      Ala     Leu      Gln




Sequence     mRNA
                            A U G    A A G      U U U       G G U    G C A   U U G   C A A
  variant    Protein         Met         Lys     Phe         Gly      Ala     Leu      Gln


            Silent DNA sequence polymorphism




                                                                                               13
        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




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




      Functional protein                     Functional protein




                                                                  14
                        Polymorphism
       • Variation in population
             – phenotype
             – genotype (DNA sequence polymorphism)
       • Variant allele > 1%
Common usage:
                              < 1%                 > 1%

                         Rare or “private”
      “Normal”                                polymorphism
                         polymorphism

       Disease                disease             ??
                                             Factor V R506Q: thrombosis,
                                             3% allele frequency




                             Mutations
                 Normal       THE BIG RED DOG RAN OUT.
                Missense      THE BIG RAD DOG RAN OUT.
                Nonsense      THE BIG RED.
   Frameshift (deletion)      THE BRE DDO GRA.
Frameshift     (insertion)    THE BIG RED ZDO GRA.



 Point mutation: a change in a single base pair




                                                                           15
                   Silent Sequence Variants

                       mRNA
    Normal                         A U G    A A G      U U U   G G C   G C A   U U G   C A A
                       Protein      Met       Lys       Phe     Gly     Ala     Leu     Gln




     Sequence          mRNA
                                   A U G    A A G      U U U   G G U   G C A   U U G   C A A
       variant         Protein      Met       Lys       Phe     Gly     Ala     Leu     Gln


   Sequence variant: a base pair change that does not change the
         amino acid sequence (a type of polymorphism)
Adapted from Campbell NA (ed). Biology, 2nd ed, 1990




                        Missense Mutations

                       mRNA
    Normal                         A U G    A A G      U U U   G G C   G C A   U U G   C A A
                       Protein      Met       Lys       Phe     Gly     Ala     Leu     Gln




                       mRNA
                                   A U G    A A G      U U U   A G C   G C A   U U G   C A A
   Missense            Protein      Met       Lys       Phe     Ser     Ala     Leu     Gln



            Missense: changes to a codon for another amino acid
            (can be harmful mutation or neutral polymorphism)
Adapted from Campbell NA (ed). Biology, 2nd ed, 1990




                                                                                               16
                        Nonsense Mutations
                       mRNA
    Normal                         A U G    A A G      U U U   G G C   G C A   U U G   C A A
                       Protein      Met       Lys       Phe     Gly     Ala     Leu     Gln




                       mRNA
                                   A U G    U A G      U U U   G G C   G C A   U U G   C A A
    Nonsense           Protein      Met



              Nonsense: change from an amino acid codon to a stop
                     codon, producing a shortened protein
Adapted from Campbell NA (ed). Biology, 2nd ed, 1990




                       Frameshift Mutations

                       mRNA
    Normal                         A U G    A A G      U U U   G G C   G C A   U U G   C A A
                       Protein      Met       Lys       Phe     Gly     Ala     Leu     Gln


                                                          U
                       mRNA
   Frameshift                      A U G    A A G      U U G   G C G   C A U   U G C   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 Campbell NA (ed). Biology, 2nd ed, 1990




                                                                                               17
                   Splice-Site Mutations
          Exon 1     Intron     Exon 2      Intron    Exon 3


                                   Exon 2


                      Exon 1                     Exon 3
  Altered mRNA


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
   •   Chromosomal translocations or inversions




                                                                      18
                      Types of Mutations
       • Point Mutations            • Transcription
          –   Silent
          –   Missense              • RNA Processing
          –   Nonsense                 – splicing
          –   (frameshift)             – poly A
                                       – RNA stability
       • Deletion/Insertion
          – small
                                    • Protein level
          – large                      – processing
                                       – stability
       • Rearrangement                 – 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.
   –   SSCP
   –   DGGE
   –   CSGE
   –   ASO
   – Chip technology
• methods for gene transfer and the power of transgenics




                                                                19
  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




      Single Strand Conformational
         Polymorphism (SSCP)
      Normal     Mutated       • DNA is denatured into
DNA                              single strands
                               • Single strands fold; shape
                                 is altered by mutations
                               • Mobility of mutant and
                                 normal strands differ in
                                 gel



Gel                        mutation




                                                              20
                   Evaluating SSCP
                                Cons
  Pros
                                • Subsequent DNA sequencing
  • Rapid, simple, and widely     needed to characterize mutation
    available for many genes
                                • Sensitivity drops with longer
  • Detects 60%−95% of            DNA sequences
    mutations in short DNA
    strands




             Denaturing Gradient Gel
             Electrophoresis (DGGE)
       Normal        Mutated    • DNA denatured into single
                                  strands
DNA
                                • Single strands reanneal into
                                  normal and mutant
                                  homoduplexes and
                                  heteroduplexes

                                • Hetero- and homoduplexes
                                  denature at different points in
                                  gradient gel


      Denaturing gradient gel




                                                                    21
       Denaturing Gradient Gel
                                 BRCA1 mutation carrier
                                1 normal homoduplex band
                                2 heteroduplex bands
                                1 mutant homoduplex band




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




                                                           22
       Heteroduplex Analysis (CSGE)

Amplify and                           Cold
 denature       Single-strand DNA
  DNA



                                                   Reannealed DNA

                                   Mutated bands

                                   Normal band




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




                                                                    23
              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




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




                                                                       24
          Evaluating ASO Analysis
                                   Cons
   Pros
                                   • Each ASO probe detects
   • Sensitive method to detect      only one specific sequence
     known mutations
                                   • Most useful for small
   • Panels of ASO probes useful     sequence changes
     to detect common mutations




     Principle of Microarray (Chip)
  Prehybridization
                   Assay Posthybridization




Synthetic DNA probes                        Probes with
                                          hybridized DNA




                                                                  25
      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
                                                             CULTURED ES CELLS
                                                            WITH TARGETED GENE
                                                                    ALTERATION
                                                                                               REMOVE BLASTOCYSTS
           REMOVE FERTILIZED OOCYTES FROM                                                      FROM PREGNANT MOUSE
            OVULATING MOUSE IMMEDIATELY                                                        FOUR DAYS
                AFTER FERTILIZATION                                                            AFTER OVULATION
          HOLDING
          PIPETTE                FEMALE PRONUCLEUS



                                                                 INJECT ES CELLS
                                                               INTO BLASTOCYST
                                     INJECTION NEEDLE
                        OOCYTE       IMPALING MALE
                                     PRONUCLEUS OF OOCYTE
                                     AND INJECTING DNA
                                                                       REIMPLANT SEVERAL BLASTOCYSTS
                   REIMPLANT SEVERAL OOCYTES                                  IN FOSTER MOTHER
                        IN FOSTER MOTHER




                                                                                       BIRTH
                            BIRTH


                                           B
               A


                                               D
                    C


             SOUTHERN BLOT
               OF TAIL DNA             NORTHERN BLOT                                   +
               A B C D                  A B C D

                                                                                       BIRTH



                                                                                               B
                                                                            A
                             BREEDING
                                                                                                   D
                                                                                   C
                                 C                                                 SOUTHERN BLOT
                                                                                     OF TAIL DNA
                                                                                    A B C D

                                                                                                       NORMAL GENE

                                                                                                       ALTERED GENE




                                                    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.




                                                                                                                      27

								
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