FEXCopyright The McGraw-Hill Companies, Inc. Permis sion required for reproduction or display(a)IntronIntronEukaryoticDNATranscriptionofeukaryoticDNAgeneratesprecursormRNA.PrecursormRNASplicingremovestheintronsfromtheprecursormRNAgenerating thefinal mRNAfortranslation.mRNA(withineukaryoticcells)mRNAextracted(b)fromcellsmRNA(isolated)ReversetranscriptasesynthesizesDNAfrommRNASingle strandofcDNADNApolymerasecompletesthecDNA,freefromintronsDouble-stranded11CDNA
11 Eukaryotic DNA (b) (a) Precursor mRNA Intron Intron Transcription of eukaryotic DNA generates precursor mRNA. mRNA (within eukaryotic cells) mRNA (isolated) Single strand of cDNA Double-stranded cDNA Splicing removes the introns from the precursor mRNA, generating the final mRNA for translation. mRNA extracted from cells Reverse transcriptase synthesizes DNA from mRNA. DNA polymerase completes the cDNA, free from introns. Copyright © The McGraw -Hill Companies, Inc. Permission required for reproduction or display
Methods for Analysis of DNAGel electrophoresis - separates DNA fragments basedon size- DNA samples are placed on soft agar gel andsubjectedtoan electric currentCopyright The McGraw-Hill Companies, hc. Permission required for reproduction or displayDNAforsample3VRestrictionendonucleasesselectivelycleavesitesofDNARestrictionfragmentsofvarying size345?(-)(-)WellsWellsSize ladderGel(+)(+)DNAmigrates12towardpositive(a)electrode
12 Methods for Analysis of DNA • Gel electrophoresis - separates DNA fragments based on size – DNA samples are placed on soft agar gel and subjected to an electric current Restriction endonucleases selectively cleave sites of DNA Restriction fragments of varying size Gel DNA migrates toward positive electrode 1 (–) 2 3 4 5 Wells (+) (a) Size ladder 1 (–) 2 3 4 5 Wells (+) DNA for sample 3 Copyright © The McGraw -Hill Companies, Inc. Permission required for reproduction or display
MethodsforAnalysisofDNA- Negative charge of molecule causes DNA to movetoward positive pole- Rate of movement is dependent on size of fragment -largerfragmentsmove more slowlyCopyright The McGraw-Hill Companies, inc.Permission required for reproduction or displayDNAforsample3RestrictionendonucleasesselectivelycleavesitesofDNARestrictionfragmentsofvarying sizeWellsWellsSize ladderGel(+)(+)13DNAmigratestowardpositive(a)electrode
13 Methods for Analysis of DNA – Negative charge of molecule causes DNA to move toward positive pole – Rate of movement is dependent on size of fragment – larger fragments move more slowly Restriction endonucleases selectively cleave sites of DNA Restriction fragments of varying size Gel DNA migrates toward positive electrode 1 (–) 2 3 4 5 Wells (+) (a) Size ladder 1 (–) 2 3 4 5 Wells (+) DNA for sample 3 Copyright © The McGraw -Hill Companies, Inc. Permission required for reproduction or display
MethodsforAnalysisofDNA-Fragments arestainedforobservation- Useful in characterizing DNA fragments and comparingfor genetic similaritiesCopyright The McGraw-Hill Companies, Inc. Permis sion required for reproduction or display.Result, following developmentesesesadesumouyNs72345WellNo.ofbasepairs inband三31602910276022605101260101014Kathy Park Talaro(b)
14 Methods for Analysis of DNA – Fragments are stained for observation – Useful in characterizing DNA fragments and comparing for genetic similarities Well 1 2 5160 3 4 5 No. of base pairs in band 5035 4910 3160 2910 2760 2260 1510 1260 1010 Result, following development © Kathy Park Talaro Sample Sample Sample Sample Known DNA Size Markers Copyright © The McGraw -Hill Companies, Inc. Permission required for reproduction or display. (b)
TheMcGraw-Hill CompaniesConcept Check:DNA fragments move through the gel duringelectrophoresis accordingtotheirA.SizeB.ChargeC.SequenceD.Orientation
Concept Check: DNA fragments move through the gel during electrophoresis according to their A. Size B. Charge C. Sequence D. Orientation