Isolation of DNA

BT 201

Biotechnology Techniques I

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DNA

• Deoxyribonucleic acid• Genetic material of all living things• Makes up chromosomes of living things

– Contained within a nucleus in eukaryotes– Found in a nucleoid region in prokaryotes– Has many regions known as genes

• Also present in mitochondria of eukaryotes• Plasmid DNA is found in prokaryotes, and

is not a part of the chromosome

Sister chromatids

CentromereT

EM

36,

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DNA

• Made up of nucleotides bonded to one another• Nucleotides that make up DNA are designated

by letters: A, C, T, G• The way the letters are arranged in DNA is its

sequence• All organisms use the same letters in their DNA• The sequence is what makes the DNA unique in

each organism, but some organisms share similar regions of sequences

DNA Structure

• DNA is a double stranded helix with nucleic acid bases in the middle and a sugar-phosphate backbone on the outside

• The two strands of DNA are held together by hydrogen bonding between the bases in a process called base pairing

• DNA strands pair in a complementary fashion, A with T and C with G

Hydrogen bond

Basepair

Ribbon model Partial chemical structure Computer model

G C

T A

A T

TA

C

C

G

G

GC

T

T

T

T

A

A

A

A

G C

A T

A

C

T

G

CG

AT

Genomic DNA

• DNA that makes up the chromosomes of living things is called genomic DNA

• Genomic DNA is responsible for an organism’s physical characteristics

• Genomic DNA results from a combination of DNA passed down from both parents in sexually reproducing organisms

• Genomic DNA is clonally reproduced in asexually reproducing organisms

• Mitochondrial DNA is found in the mitochondria of eukaryotes, and is passed down from the mother only

• Mitochondrial DNA is not part of genomic DNA

Plasmid DNA

• Not a part of chromosomal DNA

• Usually double-stranded and circular

• Most often found in bacteria, but can be found in some eukaryotes

• Can be passed from one microorganism to another

• Can carry genes like antibiotic resistance

DNA Isolation

• All types of DNA can be extracted and isolated from living organisms

• Many procedures exist for extracting and isolating DNA

• The protocol that is used depends on the type of DNA being extracted, but the overall process is the same

General Steps in DNA Isolation

• Cells must be broken open for DNA to be released

• DNA degrading enzymes must be deactivated• Membrane lipids must be removed• Proteins bound to DNA must be released from

the DNA• DNA must be precipitated in alcohol solution,

then washed, dried, and resuspended in buffer or sterile water

DNA Isolation

• Reagents used in DNA isolation can be purchased as commercially prepared kits or made up in the laboratory

• Ingredients in the commercially prepared reagents are normally proprietary information but usually contain similar chemicals to those that are made up in the lab

• Cost, effectiveness, and ease of use may be factors in determining which method is used in a particular lab

• Type of analysis that will be done with the extracted DNA is also a consideration