Download 20-DNA-technology

Biotechnology
DNA technology
• DNA diagnostics
• DNA therapy
DNA isolation
•
•
•
•
Cell lysis
Removal of proteins
DNA precipitation by ethanol
DNA dilution in water or buffer
DNA diagnostics
– PCR (amplifying part of DNA up to 106 copies)
– sequencing, alignment of all nucleotides
– restriction digestion, testing specific nucleotides
– reverse transcription – cDNA – qPCR
– blotting –
Southern (DNA)
Northern (RNA)
Western (protein)
PCR
Polymerase Chain Reaction
1.
2.
3.
PCR cycle
PCR cycling (30 cycles)
Cycle:
1. n=21
2. n=22
n = number of gene copies
(exponential growing)
3. n=23
DNA Visualization and Separation
Gel
100
50
0
1.
3.
čtvrt. čtvrt.
Východ
Západ
Sever
sieve structure of
polymer molecules with
pores
Fluorescence dye
binding to DNA and
excites photons
under UV-exposure
DNA Visualization and Separation
Gel Electrophoresis
• the movement of charged
molecules in electric field
• the movement direction
from – to +
• DNA-rate in gel depends
on DNA-fragment length
in indirect proportion
result under UV-light
Sequencing
Genome sequencing
lines in vertical
sequence gel:
Sequencing
Restriction digestion
Bacterial restriction enzymes
recognize
palindromic sequence in DNA.
Restriction digestion
Detection of mutation
 mutative DNA –
digestion
(two small fragments)
 normal DNA –
non- digestion
(one large fragment)
results after gel electrophoresis
read under UV-light
Complementary DNA (cDNA)
of eukaryotic organisms
• in laboratory, it results from
reverse transcription
• in certain gene:
cDNA < DNA
• It is quantified by qPCR
- marker of gene expression
CELL DIFFERENTIATION:
• arises because cells make and accumulate
different sets of RNA and protein molecules
• that is, they express different genes
DNA of all cells in the body of one individual
is identical ! ! !
Blotting:
transfer a substance from gel to membrane
Mechanisms:
 Capillary attraction
 electrophoretic transfer
Blotting: a basic molecular biology technique originally
created by Edwin Southern (1975)
for locating gene specific sequences on DNA fragments.
SOUTHERN BLOTTING: is used to locate and identify genes on DNA.
DNA restriction fragments are electrophoretically separated.
The fragments are blotted onto membranes, where the DNA bonds.
Hybridization with labeled DNA probes & localizing target DNAs.
NORTHERN BLOTTING: a variation on Southern blotting.
RNAs are separated by electrophoresis, transferred to membranes,
and probed with a labeled DNA probes.
WESTERN BLOTTING: another variation on Southern blotting.
Proteins are separated by electrophoresis, transferred to membranes,
and probed with an antibody that binds to the protein you are
interested in locating.
DNA therapy
Bacteria or yeast produce human proteins
coding by human genes:
- coagulation factor VIII
- insulin
- growth hormone
DNA cloning
Cloning organisms
• Reproductive
• Therapeutic
Literature
Biology, eighth edition,
Campbell, Reece
Unit three: Genetics
Chapter 20: Biotechnology
Pages 396 – 425