Biotechnology. DNA technology DNA diagnostics DNA therapy

BiotechnologyBiotechnology

DNA technologyDNA technology

• DNA diagnostics

• DNA therapy

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 PCR Polymerase Chain ReactionPolymerase Chain Reaction

1.

2.

3.

PCR cyclePCR cycle

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

SequencingSequencing

lines in vertical sequence gel:

Genome sequencing

SequencingSequencing

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

DNA cloningDNA cloning

Cloning organismsCloning organisms

• Reproductive

• Therapeutic

LiteratureLiterature

Biology, eighth edition,

Campbell, Reece

Unit three: Genetics

Chapter 20: Biotechnology

Pages 396 – 425