DNA EXTRACTION

POLYMERASE CHAIN REACTION

GEL ELECTROPHORESIS
HANNA BETH B. TANDAYAG
Instructor
Elec 2 (Fish Genetics & Breeding
The Central Dogma
Structure of DNA
 DNA EXTRACTION
• A routine procedure used to isolate DNA from the nucleus
of cells.

• A fundamental step in many biological and medical

research applications, such as genetic analysis, forensics,
diagnosis of diseases, and development of genetically
modified organisms.

• The process of DNA extraction involves breaking open cells

or tissues to release the DNA molecules from the nucleus,
which is then purified and concentrated.
 DNA EXTRACTION
To isolate DNA, we have to break down both cell and nuclear
membranes. Then we have to remove cell debris. The final
step will be to precipitate and purify DNA. DNA extraction
consists of three stages:

1. Breakdown (or so-called lysis) of cell and nuclear membrane that is

achieved by adding detergents

2. Removal of cell debris and proteins is done by adding proteases.

Proteases are enzymes that digest proteins

3. DNA purification and precipitation are done by adding ice-cold alcohol

ethanol or isopropanol.
DNA EXTRACTION

• DNA precipitate

When an ice-cold alcohol is

added to a solution of DNA, the
DNA precipitates out of the
solution and if there is enough
DNA in the solution, you may see
a stringy white mass
DNA EXTRACTION STEPS
 DNA EXTRACTION
✓ The DNA sample can now be further purified (cleaned). It
is then resuspended in a slightly alkaline buffer and
ready to use

✓ It is important to know the concentration and quality of

the DNA.

✓ Optical density readings taken by a spectrophotometer can

be used to determine the concentration and purity of DNA
in a sample. Alternatively, gel electrophoresis can be
used to show the presence of DNA in your sample and give
an indication of its quality.
DNA Replication and DNA Polymerase
 Polymerase Chain Reaction - PCR
• PCR amplifies DNA
– Makes lots and lots of copies of a few copies of
DNA
– Can copy different lengths of DNA, doesn’t have to
copy the whole length of a DNA molecule
• One gene
• Several genes
• Lots of genes
• Artificial process which imitates natural DNA
replication
 Polymerase Chain Reaction - PCR
• Sometimes called "molecular photocopying," the
polymerase chain reaction (PCR) is a fast and
inexpensive technique used to "amplify" - copy -
small segments of DNA. Because significant
amounts of a sample of DNA are necessary for
molecular and genetic analyses, studies of isolated
pieces of DNA are nearly impossible without PCR
amplification.
PCR revolutionized the study of DNA to such an
extent that its creator, Kary B. Mullis, was
awarded the Nobel Prize for Chemistry in 1993.
 Polymerase Chain Reaction - PCR
• Once amplified, the DNA produced by PCR
can be used in many different laboratory
procedures.
Ex. Most mapping techniques in the Human Genome Project
(HGP) relied on PCR.

PCR is also valuable in a number of laboratory and

clinical techniques:
1. DNA fingerprinting,
2. detection of bacteria or viruses (particularly AIDS)
3. diagnosis of genetic disorders.
 How PCR Works
• Reagents Needed
– DNA sample which you want to amplify
– DNA polymerase
• Taq DNA polymerase – Works at high temperature
– Nucleotides
• Called dNTPs
– Pair of primers
• One primer binds to the 5’ end of one of the DNA strands
• The other primer binds to the 3’ end of the anti-parallel DNA
strand
• Delineate the region of DNA you want amplified
– Water
– Buffer
 How PCR Works
• Protocol
– Put all reagents into a PCR tube
– Break the DNA ladder down the middle to create two
strands, a 5’ to 3’ strand and a 3’ to 5’ strand
• Melting or heat denaturation
– Bind each primer to its appropriate strand
• 5’ primer to the 5’ to 3’ strand
• 3’ primer to the 3’ to 5’ strand
– Annealing
– Copy each strand
• DNA polymerase
– Extending
 How PCR Works
• Temperature Protocol
– Initial Melt: 94ºC for 2 minutes
– Melt: 94ºC for 30 seconds
– Anneal: 55ºC for 30 seconds 30-35
cycles
– Extend: 72ºC for 1 minute
– Final Extension: 72ºC for 6 minutes
– Hold: 4ºC
How PCR Works
 Gel Electrophoresis of DNA
• Gel electrophoresis detects the presence of DNA
in a sample
• Gel electrophoresis detects the number of
nucleotides in a fragment of DNA
– e.g., the number of nucleotides in a DNA region which was
amplified by PCR
– Is a rough estimate, is not exact, need more sophisticated
sequencing techniques to get an exact number of
nucleotides
– Can be used to tentatively identify a gene because we
know the number of nucleotides in many genes
 How Gel Electrophoresis of DNA Works
• A sample which contains fragments of DNA is forced by an
electrical current through a firm gel which is really a sieve with
small holes of a fixed size
– Phosphate group in DNA is negatively charged so it is moved
towards a positive electrode by the current
– Longer fragments have more nucleotides
• So have a larger molecular weight
• So are bigger in size
• So aren’t able to pass through the small holes in the gel and get
hung up at the beginning of the gel
– Shorter fragments are able to pass through and move farther along
the gel
– Fragments of intermediate length travel to about the middle of the
gel
• DNA fragments are then visualized in the gel with a special dye
• The number of nucleotides are then estimated by comparing it to a
known sample of DNA fragments which is run through the gel at the
same time
 How Gel Electrophoresis of DNA Works
• Reagents Needed
– Sample of DNA fragments
– Known sample of DNA fragments
• DNA ladder
– Gel
• Agarose
– Dye to visualize the movement of the sample as it is
traveling through the gel
• Loading dye – Blue juice
• So know when to stop so sample doesn’t just run out of
the gel
– Dye to visualize DNA after it has traveled to its final
spot in the gel
• Syber® Safe
– Buffer
 How Gel Electrophoresis of DNA Works
• Equipment Needed
– Box to hold the gel
– Comb to create small wells in the agarose gel
to put the DNA sample into at the beginning of
the gel
– Positive and negative electrodes to create the
electrical current
– Power supply
– Gel photo imaging system
How Gel Electrophoresis of DNA Works