Molecular Biotechnology-Lession 3: Basic techniques in DNA technology ppt

Molecular Biotechnology
Tran Ngoc Duc, PhD
VIETNAM NATIONAL UNIVERSITY AT HO CHI MINH CITY
INTERNATIONAL UNIVERSITY
Basic techniques in DNA
technology
1. Polymerase chain reaction (PCR)
2. Gel electrophoresis
3. Primer Design
4. RT-PCR
5. Digestion
6. Southern blot/Northern blot
7. Detectable signal based techniques
8. Site-directed mutagenesis

Polymerase chain reaction
1. PCR and Primer Design

Developed in 1983 by Kary Mullis

Polymerase Chain Reaction is widely held as one of
the most important inventions of the 20th century in
molecular biology. Small amounts of the genetic
material can now be amplified to be able to a identify,
manipulate DNA, detect infectious organisms, including
the viruses that cause AIDS, hepatitis, tuberculosis,
detect genetic variations, including mutations, in human
genes and numerous other tasks.

Components in PCR reaction


Template DNA

Primers

dNTPs

Taq DNA polymerase

Buffer

Step 1: Denature DNA, 95
o
C, 5min

Step 2: Annealing:
- Denature DNA, 95
0
C, 30s-1min
- Annealing, 55-60
o
C, 1min
Step 3: Extension, 70-72
o
C, 30s-1min
Go to step 2, 32 cycles

Step 4: Holding, 4
o
C, 0s


Steps programmed in PCR

Exponentially amplifying a single or some piece of
DNA generating thousands of million copies of DNA

Total copies = 2
n
where n is the number of cycles to
copy DNA



So basically it is the cycles of heating and
cooling (thermal cycling)

Applications of PCR

DNA cloning

DNA based phylogeny

Functional analysis of gene

Hereditary diseases

Forensic sciences

Infectious diseases
2. Gel electrophoresis


A method for separating mixture of charged molecules
such as DNA, RNA or proteins under an electric field when
an electric field is applied to them

The gel acts like a sieve for separation of molecules
according to their sizes and electric charges

Gel is made of agarose for separating large molecules of
DNA, and of acrylamide for protein separation or small
DNA or RNA

Proteins, unlike nucleic acids, can have varying charges
and complex shapes, therefore they may not migrate into
the polyacrylamide gel at similar rates, or at all, when
placing a negative to positive on the sample.

In the case of nucleic acids, the direction of migration,
from negative to positive electrodes, is due to the
naturally-occurring negative charge carried by their sugar-
phosphate backbone

After the electrophoresis is
complete, the molecules in the gel
can be stained to make them visible.
Ethidium bromide, silver, or
Coomassie Brilliant Blue dye may be
used for this process.


If the molecules to be
separated contain radioactivity
added for visibility, an
autoradiogram can be recorded
of the gel

Protein electrophoresis
3. Primer design

Step 1: Denature DNA, 95
o
C, 5min

Step 2: Annealing:
- Denature DNA, 95
0
C, 30s-1min
- Annealing, 55-60
o
C, 1min

Step 3: Extension, 70-72
o
C, 30s-1min
Go to step 2, 32 cycles


Step 4: Holding, 4
o
C, 0s

Steps programmed in PCR

Primers (short DNA fragments) containing sequences
complementary to the target region along with a DNA
polymerase

Pair of primers should have similar annealing Ta,
which is usually 4-5
0
C below Tm

One primer is complementary to one end strand of DNA

Primer length usually 18-24bp, shouldn’t be over 30

GC, 40-60%

Ta = (2AT+ 4GC) - 4

Primer shouldn’t form hairpin or loop by themselves

Ta of the 2 primers shouldn’t be big different
ATGCCCTCCACTTCCGGTGCGTCGCCCTTCCTCCCAGCAGCGCCAGCACTTGCCAG
GCGGTGCAGTCGCGGCCCCAACGGCAGCAGCAGGCGGTGCAGCAGGGCGGTGCCAG
GGCCTGCGCTGGAGGGGCGGAGCGCTGGAGGCAGCGTACGAGCGGTGTGGCGCGGT

GTGCAAGGAGTACGCCAAGACCTTCTACCTGGGCACGCAGCTCATGACCCCCGTCC
AGGCACGCTGCATCTGGGCCATCTACGTGTGGTGCAGGCGCACGGATGAGCTGGTG
GATGGCCCTAATGCCTCCAAGATCACACCACAGGCCCTGGACCGGTGGGAGGAGCG
F:?
R:?
Ta=?
Ta=?

/>
The melting temperature (T
m
) is the temperature at which one-
half of a particular DNA duplex will dissociate and become
single strand DNA

The actual (T
m
) is influenced by the concentration of Mg
2+
, K
+
,
and co-solvents. There are numerous computer programs to
assist in primer design.
4. RT-PCR

Reverse transcription polymerase chain reaction (RT-
PCR) is a variant of polymerase chain reaction (PCR)
mRNA
DNA

cDNA
Reverse transcriptase
Taq DNA polymerase

In RT-PCR, an RNA strand is first reverse transcribed into
its DNA complement (complementary DNA, or cDNA) using
the enzyme reverse transcriptase