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Human Genomic DNA Isolation. Zelha Nil Nov 2009. DNA Structure. Composed of nucleotides : A, T, G, C Synthesized in 5’ to 3’ direction through formation of phosphodiester bonds betw deoxyribose & phosphate : Sugar - Phosphate backbone

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human genomic dna isolation

Human Genomic DNA Isolation

Zelha Nil

Nov 2009

dna structure
DNA Structure
  • Composed of nucleotides: A, T, G, C
  • Synthesized in 5’ to 3’ directionthroughformation of phosphodiesterbondsbetwdeoxyribose & phosphate: Sugar-Phosphatebackbone
  • Doublehelix: H-bondingbetwcomplementarybases
  • Specificsequence of bases: protein structure & geneticinheritance
organization of human genome
Organization of Human Genome
  • Human genome: Total genetic information (DNA content) in human cells
    • Nuclear genome: 99.9995% of the total genetic information
    • Mitochondrial genome: the remaining 0.0005%
  • Nuclear genome
    • Chains of DNA organized into chromosomes
    • Human: 3x109 bp packed into 23 chromosomes, 2n=46
    • Human chromosomes: 50-250 Mb in length
    • Each chromosome: Single long molecule of DNA



One from each parent: non-identical


Sister chromatids



Euchromatin: Lightly packed form of chromatin that is rich in gene concentration

  • Heterochromatin: Tightly packed form of DNA
why we isolate genomic dna
Why we isolate genomic DNA?
  • PCR; presence of sequencesoramplification of targetsequences (mutationalanalysis, cloning)
  • Southernblot; presence of sequences (DNA fingerprinting, cloning)
  • Sequenceanalysis (mutationalanalysis, sequencingthegenome)
  • DNA fragmentation; indication of apoptosis
  • RE digestion; RFLP (DNA polymorphisms), DNA fingerprinting, cloning, PCR
dna polymorphisms
DNA polymorphisms
  • Definition: Differences in nucleotide sequence among individuals of a species
  • Result from
    • Point mutations
    • Random indels
    • Variable repeat numbers in a repetitive locus
  • Used for DNA fingerprinting
    • Repetitive DNA sequences
    • Restriction fragment length polymorhisms (RFLP)
rflp analysis
RFLP analysis
  • Based on variability in restriction enzyme (RE) cut sites betw individuals
  • Single base changes in DNA
    • Introduce or delete a RE cutsite
    • For ex: A mutation changing the sequence AGATCC to GGATCC introduce a BamH1 site into that segment of DNA
  • Alterationin RE cut site:
    • Variation in the length of the fragments
    • Difference in the position of certain gel bands betw individuals
common procedures
Common procedures
  • Phenol-chloroform extraction (manual)
  • Salting out (our protocol)
  • ASSIGNMENT: What are the differences betw these 2 methods & which one is more efficient or advantageous? What can be other methods alternative to these? (1 page)
dna isolation by salting out method
DNA isolation by salting out method
  • Put 750 µl blood, 750 µl TKM buffer and 10 µl Triton X-100 into 2 ml eppendorf tube, mix well by inversions.
  • Centrifuge at 1000g for 10 min at RT.
  • Discard supernatant slowly, save the pellet.
  • Add 750 µl TKM buffer and resuspend the pellet.
  • Centrifuge at 1000g for 10 min at RT.
  • Repeat the steps 3-5 2 more times.
  • Resuspend the pellet in 200 µl TKM buffer.
  • Add 20 µl of 10%SDS, mix well.
  • Incubate the samples at 58oC for 10 min.
cont d
  • Add 75 µl cold saturated NaCl.
  • Centrifuge at 14000g for 10 min at 4oC.
  • Save the supernatant (300 µl) into a new 1.5 ml eppendorf tube.
  • Add 2x volume (600 µl) of absolute ethanol, invert slowly several times. DNA is visible in this step.
  • Incubate the samples at -20oC for 30 min.
  • Centrifuge at 10000g for 10 min at 4oC.
  • Pour off the ethanol, let the eppendorfs dry under hood.
  • Add 200 µl TE buffer pH:8.0, resuspend.
  • Incubate at 37oC for at least 2 hours.

TKM buffer (TrisHCl, EDTA)

Hypotonicbufferforlysis of RBC and WBC enhancedbyinversions.

  • Triton X 100, SDS (10% w/v)


  • SaturatedNaCl


  • Absoluteethanol (96-100% v/v)

Added 2-3 volumes of thesolutiontocollect DNA (orprecipitatenucleicacids) fromaquousphases since nucleicacidstendtoinsolubilize in ethanol.

  • TE buffer (10mM Tris, 1mM EDTA, pH 7.5)

Dissolve DNA andstoragebuffer