1. DNA Analysis Techniques

2. DNA analysis: Fundamental 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

3. Isolation of Nucleic Acids
Goals:
Removal of proteins
DNA vs RNA
Isolate specific type of nucleic acid

4. DNA isolation

5. Isolation of DNA
A good prep should:
Not contain cellular proteins
Not contain RNA
Be of high molecular weight

6. 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

7. Gel electrophoresis

8. 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.

9. Basic Principle of Electrophoresis
A mixture of DNA molecules becomes organized by size

10. 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”

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

12. 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

13. A Typical Image of an Agarose Gel Under UV Light
Decreasing
DNA
Size
Largest DNA fragments
Smallest DNA fragments
The DNA fragments can be visualized using a special dye that specifically binds DNA and fluoresces under illumination with UV light

14. The electrophoretic migration rate of DNA through agarose gel depends on the following parameters:
The size of the DNA molecules
DNA molecules travel through gel at a rate inversely proportional to the logarithm of their molecular weight or number of base pairs.
The concentration of agarose
A DNA fragment of a given size migrates at different rates in gels containing different concentrations of agarose
The voltage applied
Normally the migration rate of DNA fragments is directly proportional to the voltage applied
The conformation of the DNA
The buffer used for electrophoresis

15. Effects of Changing Gel Concentration
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

16. Effect of Percent Agarose on Fragment Separation: An example
The 1kbp ladder and the 100bp ladder were run with 0.8 and 1.6 % agarose gels under the same conditions
1600
1000
1600 bp
500
400 bp
1000 bp
300 bp
500 bp
200 bp
0.8% Agarose
80V – 2 hours
1.6% Agarose
80V – 2 hours

17. 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 less
DNA when compared to the lower
band. The intensity of the bands
are proportional to the amount of DNA at that position in the gel

18. 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

19. A Typical Electrophoresis Gel Setup
Negative end
Direction
of DNA
movement
Positive end

20. 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

21. 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)

22. DNA Ladders

23. 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

24. 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

25. Thanks