DNA Analysis Techniques

DNA analysis: Fundamental techniques

• Techniques– Gel electrophoresis – Restriction analysis– DNA hybridization– DNA sequencing– RFLP (Restriction fragment length polymorphism)

• Applications of rDNA Technology– Basic research– Medical applications– Forensic applications– Agriculture applications– Environmental applications

Isolation of Nucleic Acids

• Goals:– Removal of proteins– DNA vs RNA– Isolate specific type of nucleic acid

DNA isolation

Isolation of DNA

• A good prep should:

– Not contain cellular proteins

– Not contain RNA

– Be of high molecular weight

Quality and Quantity of DNA

• Gel electrophoresis

– Quantity: Band intensity is semi-quantitative

– Quality: Look for high MW DNA single clear band

• Sheared DNA indicates poor quality smear

Gel electrophoresis

What is Gel Electrophoresis?

• Electro = flow of electricity, phoresis, from the Greek = to carry across

• A gel is a colloid, a suspension of tiny particles in a medium, occurring in a solid form, like gelatin

• Gel electrophoresis refers to the separation of charged particles located in a gel when an electric current is applied

– Charged particles can include DNA, amino acids, peptides, etc

• Agarose gel electrophoresis is routinely used for the preparation and analysis of DNA.

• Gel electrophoresis is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field.

Basic Principle of Electrophoresis

A mixture of DNA molecules becomes organized by size

How does gel electrophoresis separate DNA fragments?

• The gel acts as a sieve to filter the DNA fragments

• The DNA fragments are naturally negatively charged due to the phosphate backbone

• DNA fragments of differing sizes will move though the gel at differing rates

• Smaller fragments move faster through the gel and larger fragments move slower

• The electrostatic charges set up in the gel act as the “force”

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

+ + + + + + + + + + + + + + + + + + + + + + + + ++ + + + + + + + + +

DNA is negatively charged and therefore repelled from the negative pole and attracted towards the

positive pole

Gel staining using Ethidium Bromide

The most rapid, sensitive, and reproducible method for staining s- and ds DNA

Binds to ds nucleic acid by intercalation between stacked base pairs

The intensity of fluorescence (In UV) of dye bound to DNA is much greater than that of free dye suspended in agarose.

Permits direct observation of the progress of electrophoresis

A Typical Image of an Agarose Gel Under UV Light

• The DNA fragments can be visualized using a special dye that specifically binds DNA and fluoresces under illumination with UV light

Decreasing DNASize

Largest DNA fragments

Smallest DNA fragments

The electrophoretic migration rate of DNA through agarose gel depends on the following parameters:

The size of the DNA moleculesDNA molecules travel through gel at a rate inversely

proportional to the logarithm of their molecular weight or number of base pairs.

The concentration of agaroseA DNA fragment of a given size migrates at different

rates in gels containing different concentrations of agarose

The voltage appliedNormally the migration rate of DNA fragments is directly proportional to the voltage applied

The conformation of the DNA

The buffer used for electrophoresis

By increasing the agarose concentration the smaller DNA fragments will give a clearer separation

By lowering the agarose concentration the larger fragments of DNA will give a clearer separation

By optimizing the % agarose one can clearly separate a mixture of similar DNA fragments

Effects of Changing Gel Concentration

Effect of Percent Agarose on Fragment Separation: An example

1.6% Agarose

80V – 2 hours

0.8% Agarose

80V – 2 hours

1600 bp

1000 bp 500 bp 200 bp

1600

1000

500300 bp400 bp

The 1kbp ladder and the 100bp ladder were run with 0.8 and 1.6 % agarose gels under the same conditions

The Intensity of the Band is Proportional to the Concentration Of DNA

• An important point to remember is that the intensity of the band is proportional to the amount of DNA found in the band

The upper band has far lessDNA when compared to the lowerband. The intensity of the bands are proportional to the amount of DNA at that position in the gel

Assessing the Integrity of DNA

High Quality Genomic DNA

>95% DNA will be of high molecular weight, migrating as intact band near the top of the gel

Very little evidence of smaller fragments indicated by a smear of many different sized DNA fragments

A Typical Electrophoresis Gel Setup

Positive end + + + + + + + + + +

Negative end - - - - - - - - - - - - -

Direction

of DNA

movement

Ladder

• A ladder is a mixture of DNA fragments of selected sizes

• When run in a gel electrophoresis, these fragments will separate into distinct bands that can be used as references

• The size of a fragment is always stated as [X] base pairs (bp)

• Two common ladders are the 100 bp and the 1 kbp (1000 bp) ladders

Typical Ladders-100 bp & 1 kbp (1000 bp)

• The 100 bp ladder is composed of a mixture of small fragments (100 to 3000 bp)

• The 1000 bp ladder is composed of a mixture of larger fragments (250 to 10,000 bp)

DNA Ladders

DNA fragments can be separated, and their sizes can be determined with the use of gel electrophoresis. The fragments can be viewed by using a dye that is specific for nucleic acids or by labeling the fragments with a radioactive or chemical tag.

Concepts

Still more….

• The DNA band of interest can be cut out of

the gel and the DNA extracted

• Or DNA can be removed from the gel by

Southern Blotting

Thanks