biotechnology
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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 10 6 copies) - PowerPoint PPT PresentationTRANSCRIPT
DNA isolationDNA isolation
• Cell lysis
• Removal of proteins
• DNA precipitation by ethanol
• DNA dilution in water or buffer
DNA diagnosticsDNA 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 cycling (30 cycles)PCR cycling (30 cycles)
1. n=21
2. n=22
3. n=23
Cycle:
n = number of gene copies (exponential growing)
DNA Visualization and SeparationDNA Visualization and Separation
0
50
100
1.čtvrt.
3.čtvrt.
Východ
Západ
Sever
Gelsieve structure of polymer molecules with pores
Fluorescence dyebinding to DNA and excites photonsunder UV-exposure
DNA Visualization and SeparationDNA 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
Restriction digestionRestriction digestion
Bacterial restriction enzymes
recognize
palindromic sequence in DNA.
Restriction digestionRestriction digestionDetection 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)Complementary DNA (cDNA)of eukaryotic organismsof 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: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 ! ! !
BlottingBlotting: : transfer a substance from gel to membranetransfer a substance from gel to membrane
Mechanisms:
Capillary attraction
electrophoretic transfer
BlottingBlotting:: a basic molecular biology technique originally a basic molecular biology technique originally created by Edwin Southern created by Edwin Southern (1975)(1975)
for locating gene specific sequences on DNA fragments. 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 therapyDNA therapy
Bacteria or yeast produce human proteins coding by human genes:
- coagulation factor VIII
- insulin
- growth hormone