Johannotes
CRASH COURSE Biochemistry 4th shifting
Molecular Genetics, Recombinant DNA, and
Genomic Technology Part 1
Short definitions
1. Recombinant DNA
- altered DNA that results from the insertion
of sequence of deoxyribonucleotides not
previously present into an existing
molecule of DNA (existing + new
sequence
2. Chimeric DNA
- type of recombinant DNA but sequences
are derived from two different species
3. Probe
- oligonucleotide use to identify aspecific
base sequence in a DNA target
4. Hybridization
- “the process” – association of
complementary strands of nucleic acids
- Perfect matches withstand high
temperature and low ionic strength buffer
5. Gel Electrophoresis
- USED IN SEPARATING DNA
-based on the movement of charged
particles in an electric field toward an
electrode of opposite charge
-Bakit negative ang DNA ? because of
their Phosphate group … ano pake natin
now ?
-In gel electrophoresis negatively charge
will move to the positive electrode …
-Smaller fragments will move FASTER.
Larger SLOWER
a. Agarose Gel Electrophoresis
- Larger fragments (thousands of
oligonucleotides)
b. Polyacrylamide Gel (PAGE)
- Smaller fragments (hundreds of
oligonucleotides)
6. Detection methods
a. Radioactive labeling 32p or 35S
- Autoradiography – after separation,
labeled oligonucleotides placed in
contact with an X-ray film
- Will show substances as dark
bands
b. Fluorescent compound
- Labeled with different colors
7. Nuclease – cleaves nucleic acids
8. Endonuclease – cleaves the internal
phosphodiester bond
9. Restriction Endonuclease PAKI
TANDAAN
- can cut DNA at highly specific sequences
within the molecule (very specific)
- presence in a given bacterium restricted
the growth of certain bacterial viruses
(bacteriophages); protect the host bacterial
DNA from foreign DNA (defense
mechanism of bacteria against viruses)
-HOW? May kasama siya lagi site specific
DNA methylase na memethylase yung
VIRAL DNA kasi si cos DNA rendering it
unsuitable substrate is UNMETHYLATED
- Recognition sequences are usually 4-6
base pairs long and palindromic
5’ GGATCC 3’
3’ CCTAGG 5’
10. Types of Cuts of Restriction
Endonucleases
a. Sticky Ends
- Leave 2-4 nucleotides of one strand
unpaired at each resulting ends
- Can base pair with each other or
with complementary sticky ends of
other DNA fragments
GAATTC G AATTC
——>
CTTAAG CTTAA G
b. Blunt Ends
- No OVERHANGS
11. Blotting Techniques
a. Southern – DNA with specific base
sequences
- Takes advantage of the property of
nitrocellulose that it tenaciously
bind to single stranded DNA
b. Northern –specific RNA
sequences
c. Western -Protein through use of
antibodies
