Human Genomic DNA Isolation

Human Genomic DNA IsolationZelha 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

• Double helix: H-bonding betw complementary bases

• Specific sequence of bases: protein structure & genetic inheritance

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

Homologous chromosomes

Sister chromatids

Replication

One from each parent: non-identical

Identical

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

•Heterochromatin: Tightly packed form of DNA

Why we isolate genomic DNA?

• PCR; presence of sequences or amplification of target PCR; presence of sequences or amplification of target sequences (mutational analysis, cloning)sequences (mutational analysis, cloning)

• Southern blot; presence of sequences (DNA Southern blot; presence of sequences (DNA fingerprinting, cloning)fingerprinting, cloning)

• Sequence analysis (mutational analysis, sequencing the Sequence analysis (mutational analysis, sequencing the genome)genome)

•DNA fragmentation; indication of apoptosisDNA fragmentation; indication of apoptosis• RE digestion; RFLP (DNA polymorphisms), DNA RE digestion; RFLP (DNA polymorphisms), DNA

fingerprinting, cloning, PCRfingerprinting, cloning, PCR

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• Based on variability in restriction enzyme (RE) cut sites betw

individuals • Single base changes in DNA ▫ Introduce or delete a RE cut site▫ For ex: A mutation changing the sequence AGATCC to GGATCC

introduce a BamH1 site into that segment of DNA• Alteration in RE cut site:▫ Variation in the length of the fragments▫ Difference in the position of certain gel bands betw individuals

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 •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) Hypotonic buffer for lysis of RBC and WBC enhanced by Hypotonic buffer for lysis of RBC and WBC enhanced by inversions.inversions.

• Triton X 100, SDS (10% w/v)Triton X 100, SDS (10% w/v)Detergents to solubilize lipids and proteins Detergents to solubilize lipids and proteins

• Saturated NaCl Saturated NaCl Nuclei lysis buffer Nuclei lysis buffer

•Absolute ethanol (96-100% v/v)Absolute ethanol (96-100% v/v)Added 2-3 volumes of the solution to collect DNA (or Added 2-3 volumes of the solution to collect DNA (or precipitate nucleic acids) from aquous phases since nucleic precipitate nucleic acids) from aquous phases since nucleic acids tend to insolubilize in ethanol.acids tend to insolubilize in ethanol.

• TE buffer (10mM Tris, 1mM EDTA, pH 7.5)TE buffer (10mM Tris, 1mM EDTA, pH 7.5)Dissolve DNA and storage bufferDissolve DNA and storage buffer