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GBE202 Bioengineering Laboratory

The document discusses various techniques for isolating and analyzing nucleic acids including DNA extraction from blood cells, gel electrophoresis to separate DNA fragments by size, PCR to amplify DNA, DNA sequencing including Sanger sequencing, and the human genome project. It also provides instructions for a nucleic acid purification experiment using buccal cells.

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Azzam Safi
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28 views23 pages

GBE202 Bioengineering Laboratory

The document discusses various techniques for isolating and analyzing nucleic acids including DNA extraction from blood cells, gel electrophoresis to separate DNA fragments by size, PCR to amplify DNA, DNA sequencing including Sanger sequencing, and the human genome project. It also provides instructions for a nucleic acid purification experiment using buccal cells.

Uploaded by

Azzam Safi
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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GBE202 Bioengineering

Laboratory
Nucleic Acid Isolation
Dr. F. Tuba Akdeniz
2024 Spring
• DNA manuplation
• DNA isolation
• Gel Electrophoresis
• PCR
DNA Extraction

• DNA from blood is isolated


from one of three types of
leukocytes: monocytes,
lymphocytes, or
granulocytes
Gel Electrophoresis Separates DNA Fragments of
Different Sizes
• After a large DNA molecule is cleaved
into smaller pieces with a restriction
nuclease, the DNA fragments can be
separated from one another on the
basis of their length by gel
electrophoresis—the same method
used to separate mixtures of proteins
• A mixture of DNA fragments is loaded
at one end of a slab of agarose or
polyacrylamide gel, which contains a
microscopic network of pores.
• When a voltage is applied across the
gel, the negatively charged DNA
fragments migrate toward the positive
electrode; larger fragments will
migrate more slowly because their
progress is impeded to a greater
extent by the gel matrix.
Gel Electrophoresis Separates DNA Fragments of
Different Sizes
DNA molecules can be separated by size using gel electrophoresis

• Schematic illustration
compares the results of
cutting the same DNA
molecule with two
different restriction
nucleases, EcoRI
(middle) and HindIII
• Restriction nucleases
cleave DNA at specific
nucleotide sequences.
DNA Cloning by PCR

• A powerful and versatile method for amplifying DNA, known


as the polymerase chain reaction (PCR), provides a more rapid
and straightforward approach to DNA cloning, particularly in
organisms whose complete genome sequence is known. Today,
most genes are cloned via PCR.
DNA Cloning by PCR
• Invented in the 1980s, PCR revolutionized the
way that DNA and RNA are analyzed. The
technique can amplify any nucleotide sequence
rapidly and selectively. Unlike the traditional
approach of cloning using vectors—which relies
on bacteria to make copies of the desired DNA
sequences—PCR is performed entirely in a test
tube.
Multiple Cycles of Amplification In Vitro Generate Billions of Copies of the
Desired Nucleotide Sequence
• PCR is an iterative process in which the cycle of amplification is
repeated dozens of times.
• At the start of each cycle, the two strands of the doublestranded
DNA template are separated and a unique primer is annealed to
each.
Multiple Cycles of Amplification In Vitro Generate Billions of Copies of the
Desired Nucleotide Sequence
DNA Cloning by PCR
Principle of PCR
Coronavirus real time RT-PCR Test
DNA Sequencing
• In 1977, Frederick Sanger, a British biochemist and two-
time Nobel Prize winner, invented a way to determine
the base sequence of a small piece of DNA.
• “Sanger sequencing” remains the conceptual basis for
techniques that today can sequence an entire human
genome in a day.
• Sanger’s method is still used to sequence individual
genes, or to check the accuracy of a selected sequenced
area of a genome.
• The ability to sequence millions of small pieces at once is
the basis of newer methods, called “next-generation
sequencing,” that can handle much larger DNA
molecules much faster.
Human Genome Project
• The use of unique sequences is why the human genome
project did not uncover copy number variants. For
example, the sequence CTACTACTA would appear only as
CTA.
• Researchers did not at first appreciate the fact that
repeats are a different form of information and source of
variation than DNA base sequences.
• A balance was necessary between using DNA pieces
large enough to be unique, but not so large that the
sequencing would take a very long time.
Human Genome Project

Annotation of a gene variant might include:


■ the normal function of the gene;
■ mode of inheritance;
■ genotype (heterozygote, homozygote,
compound heterozygote); and
■ frequency of a variant in a particular
population.
Lessons from the Human Genome Project
• The Human Genome Project, one of the most ambitious
scientific projects ever undertaken, achieved a monumental
goal: sequencing the entire human genome.
• Since its completion in 2003, this project has laid the
groundwork for thousands of scientific studies associating
genes with human diseases.
Lessons from the Human Genome
Project
• A genome, in contrast, is a complete set of DNA
instructions, including all of a person’s genes. In
humans, the genome consists of 3 billion bases.
• All humans share about 99.9% of this genome, and
the remainder is variable (and 0.1% of 3 billion is still
3 million bases – nothing to sneeze at!).
• A spot in the genome that can differ between people
(e.g., where some people have an A and others have
a G) is called a single nucleotide polymorphism, or
SNP
• The version of a SNP a person has is called their
genotype, and these small genetic differences are
part of what makes people unique.
Lessons from Human Genome Project
NUCLEIC ACID PURIFICATION
EXPERIMENT: NUCLEIC ACID PURIFICATION

Buccal Cell Collection

1- To collect buccal cells, scrape the inside of the mouth 2 minutes with a buccal Collection
Brush.

Cell Lysis

2- Dispense 300 µl Cell Lysis Solution into a 1.5 ml micro centrifuge tube. Remove the collection
brush from its handle using sterile scissors or a razor blade, and place the detached head in the tube.

3- Incubate at 65°C for 15 min.

4- Remove the collection brush head from the cell lysis solution, scraping it on the sides of the
tube to cover as much liquid as possible.

Protein Precipitation

5- Add 100 µl of Protein Precipitation solution and,

• Vortex the samples vigorously for 20 seconds to mix the protein precipitation solution
uniformly with the lysate

• İncubate 5min. on ice

6- Centrifuge at 14.000 g for 3 min. The precipitated proteins should form a tight pellet. If the
protein pellet is not tight
7- Pipette 300 µl Isopropanol and 0.5 µl Glycogen solution into a clean 1.5ml micro centrifuge
tube, and add the supernatant from the previous step by pouring carefully.

8- Mix the sample by inverting it gently 50 times.

9- Centrifuge at 14.000 g for 5 min. The DNA may or may not be visible as a small white pellet,
depending on yield.

10- Discard the supernatant and drain the tube briefly on clean absorbent paper, taking care that
the pellet remains in the tube.

11- Add 300 µl %70 Ethanol and invert the tube several times to wash the DNA pellet.

12- Centrifuge at 14.000 g for 1 min

13- Carefully discard the supernatant. İnvert and drain the tube on clean, absorbent paper and
allow to air dry for 5 min.

14- Add 100 µl of DNA Hydration Solution and vortex for 5 sec at medium speed to mix.

15- Incubate at 65°C for 1 hour to dissolve the DNA.

16- Incubate at room temperature overnight with gentle shaking. Ensure tube cap is tightly
closed to avoid leakage. Samples can then be centrifuged briefly and transferred to a storage tube.
A 260/280 ratio of ~1.8 is generally accepted as “pure” for
DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA.

• The maximum absorbance of nucleic acids occurs at a


wavelength of 260 nm
• If the ratio is appreciably lower in either case, it may indicate the
presence of protein, phenol or other contaminants
NanoDrop

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