Module 2.3- DNA Extraction & Assembling DNA Parts

DNA extraction

A process that isolates DNA from a biological sample, such as blood, tissue, or saliva, by breaking open the cells and separating the DNA from proteins and other components

Purpose of DNA extraction

• Obtain DNA from cells

• Isolate and purify DNA from contaminants such as proteins, carbohydrates, lipids or other nucleic acids

• Use the purify DNA for further studies or applications

Potential uses of purified DNA

• Genetic Engineering Plants and Animals

  • DNA with desirable genetic traits are extracted and purified and transplanted into the organism’s genome.

• Pharmaceutical Products

  • Pharmaceuticals made via recombinant genetics include the Hepatitis B vaccine and human growth hormone. In addition to a number of other hormones created using DNA extraction, the most common is insulin.

• Medical Diagnosis

  • DNA is extracted from the patient and purified for analysis to diagnose conditions such as cystic fibrosis or Huntington’s disease

• Identity Verification

  • Genetic fingerprinting is a process that matches genetic material from an individual with other genetic material available. One example is paternity testing, to determine someone’s biological father

Harvesting

• Routinely isolate DNA from human, fungal, bacterial and viral sources

• Some cells may need pretreatment, e.g.:

  • Whole blood

  • Tissue samples

• Need sufficient samples for adequate yield

3 steps of DNA extraction

Lysis, separation, and purification

Lysis

Release DNA by breaking the cell

Separation

Separate DNA from other cell components (e.g., carbohydrates, proteins)

Purification

Remove any unwanted material from DNA

Types of lysis

• Mechanical Disruption

  • Homogenization

  • Mortar and pestle

• Chemical Lysis

  • Detergents (e.g. SDS)

  • Buffering Salts (e.g. Tris-Cl, EDTA)

Methods of separation

• Protein Denaturation/Precipitation

• RNA denaturation

• DNA precipitation

• DNA adsorption

Protein denaturation/precipitation in separation

Methods include:

• Enzymes (e.g. Proteinase K)

• Phenol/Chloroform extraction

  • DNA in aqueous phase

  • Proteins in organic phase

• Chemical (e.g. Guanidine hydrochloride)

• Salting out

  • High salt concentration (e.g. LiCl, NaCl)

  • Low pH

RNA denaturation in separation

Via RNase (enzyme)

DNA precipitation in separation

Precipitate DNA using alcohol (i.e., ethanol, isopropanol)

DNA adsorption in separation

• Use solid matrix (e.g. membrane) to bind DNA

• Wash away contaminants

• Elute DNA from column

Purification method

• Wash DNA with 70% ethanol

• Re-suspend DNA in buffer

  • Usually in TE buffer (Tris-HCl and EDTA)

Methods of analyzing DNA

Gel electrophoresis and spectrophotometry

Gel electrophoresis

• Analyze DNA quality by electrophoresis

• Determine the size of DNA isolated

Spectrophotometry

• DNA concentration can be determine using absorbance at 260 nm

  • 1 𝐴260 = ~50 𝜇𝑔/𝑚𝑙

• DNA purity can be determine by A260/A280 ratio

Traditional molecular cloning workflow

1. Preparing both the cloning vector and the DNA fragment (insert) to be cloned

2. Ligating the insert into the vector to create a recombinant molecule

3. Transforming this recombinant molecule into a host organism like bacteria

4. Screening the colonies to identify the correct clone

NOTE: Must cut plasmid and gene fragments with the same restriction enzymes (Scissors)

When can we form a recombinant plasmid?

Once we have amplified (and verified) our DNA (biological parts)

What we need to form recombinant plasmid

• Amplified and purified biological part(s)

• Target plasmid

• Reagents

Techniques to form recombinant plasmid

• DNA extraction (miniprep)

• Restriction/Ligation (cut and paste)

• Gel

Plasmid

• Circular or linear double-stranded DNA

• Replicate separately from the chromosome, uses the same enzymes -

• Typically much smaller than chromosomes: 1 kbp to more than 1 Mbp. less than 5% of the size of the chromosome

• Usually not “essential” to the cell

• Each cell can contain more than one copy or even one type of plasmid

Features of a recombinant plasmid

1. Origin of replication

2. A gene that is advantageous for survival, such as an antibiotic resistance gene

3. Cloning sites

4. Promoter

5. Insert, restriction sites, ribosome binding site, primer binding sites

Origin of replication (ori)

DNA sequence that directs initiation of plasmid replication by recruiting the bacteria transcription machinery

• Controls the host range and copy number of the plasmid

• Not all origins of replication are created equal. Some will produce many plasmid copies and others produce just a few copies (5 -700)

Cloning sites

Sequences that can be cut with restriction enzymes

Ribosome binding site

Various Shine-Dalgarno sequences have been found in prokaryotic mRNAs

• These sequences lie about 6-10 nucleotides upstream from the AUG start codon.

• Activity of a RBS can be influenced by the length and nucleotide composition of the spacer separating the RBS and the initiator AUG.

• Different in prokaryotes and eukaryotes

Primer binding site

• Initiation point for PCR amplification or DNA sequencing of the plasmid

• Can be utilized to verify the sequence of the insert or other regions of the plasmid.

Why are plasmids with the same ori incompatible?

They cannot be in the same bacteria, because they will compete for the same machinery, creating an unstable and unpredictable environment

The best choice of ori depends on…

• How many plasmid copies you want to maintain

• Which host or hosts you intend to use

• Whether or not you need to consider your plasmid's compatibility with one or more other plasmids.

Promoter 

100 to 1000 base pairs long and found upstream of their target genes.

• Controls the binding of the RNA polymerase and transcription factors

• RNA pol in bacteria is a single enzyme

• Eukaryotes have multiple polymerases → the promoter must be compatible with the type of RNA to be made

What is the strength of a promoter important for?

Controlling the level of insert expression (i.e., a strong promoter directs high expression, whereas weaker promoters can direct low/endogenous expression levels)

Constitutive promoter

• A DNA sequence that drives the continuous transcription of a gene

  • The gene is expressed at a constant level regardless of external conditions or regulation.

• It is always "on" and binds RNA polymerase to initiate the production of mRNA, leading to the consistent expression of a protein.

Inducible promoter

• A DNA sequence that controls the transcription of a gene in response to a specific signal, allowing for gene expression to be turned on or off, or adjusted.

• This regulation is often achieved using activators or repressors, where a small molecule inducer can bind to the regulatory protein to either enable or block its interaction with the DNA, thereby controlling the expression of downstream genes.