Human Genomic DNA Isolation

Zelha Nil Nov 2012

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
Replication

One from each parent: non-identical

Sister chromatids

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 sequences (mutational analysis, cloning) Southern blot; presence of sequences (DNA fingerprinting, cloning) Sequence analysis (mutational analysis, sequencing the genome) DNA fragmentation; indication of apoptosis RE digestion; RFLP (DNA polymorphisms), DNA fingerprinting, 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 l blood, 750 l TKM buffer and 10 l 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 l 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 l TKM buffer. Add 20 l of 10% SDS, mix well. Incubate the samples at 58oC for 10 min.

Contd
Add 75 l cold saturated NaCl. Centrifuge at 14000g for 10 min at 4oC. Save the supernatant (300 l) into a new 1.5 ml eppendorf tube. Add 2x volume (600 l) 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 l 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 inversions.

Triton X 100, SDS (10% w/v)

Detergents to solubilize lipids and proteins

Saturated NaCl
Nuclei lysis buffer

Absolute ethanol (96-100% v/v)

Added 2-3 volumes of the solution to collect DNA (or precipitate nucleic acids) from aquous phases since nucleic acids tend to insolubilize in ethanol.

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

Dissolve DNA and storage buffer