1 / 12

Human Genomic DNA Isolation

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

parry
Download Presentation

Human Genomic DNA Isolation

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Human Genomic DNA Isolation Zelha Nil Nov 2009

  2. 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

  3. 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

  4. Homologous chromosomes One from each parent: non-identical Replication Sister chromatids Identical

  5. Euchromatin: Lightly packed form of chromatin that is rich in gene concentration • Heterochromatin: Tightly packed form of DNA

  6. 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

  7. 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)

  8. 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

  9. 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)

  10. 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.

  11. 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.

  12. TKM buffer (TrisHCl, EDTA) Hypotonicbufferforlysis of RBC and WBC enhancedbyinversions. • Triton X 100, SDS (10% w/v) Detergentstosolubilizelipidsandproteins • SaturatedNaCl Nucleilysisbuffer • 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

More Related