H11 Brock samenvatting
11.1 restriction enzymes and nucleic acid separation
In vivo = in living organisms
In vitro = in test tube.
Restriction enzymes recognise specific sequences and cut them, protect cell from other DNA. Can’t
cut own DNA because on every sequences in its own DNA hangs a methyl-group that protects the
DNA.
Mechanisms of restriction enzymes
3 classes: I, II and III. I and III bind on sequences on DNA but cut a few sequences later. II cut exactly
at the sequence.
A sequence: 4-8 bp, written from right to left and left to right the same -> palindromes.
Sticky(forms two single strand DNA ends) and blunt ends(right in half)
Modification: protection form restriction
Modification enzymes protect own DNA against restriction enzymes by adding a methyl-group at the
sequences. This modifying is called methylases.
Gel electrophoresis: Separation of DNA Molecules
Based on moleculesize. Also used to make sure that the DNA amplification was successful. Separating
charged molecules by an electrical field. Gel made of agarose for separating DNA fragments. DNA
move from negative to positive pole because DNA is negative, in this way the DNA gets separated. By
adding ethidium bromide the DNA will fluoresce under UV light.
11.2 Nucleic Acid Hybridization
Single stranded DNA can bind with other single stranded DNA or RNA , can be used in detecting,
characterizing and identifying segments of DNA. Known single stranded DNA segments that are used
are called nucleic acid probes, can be radioactive labelled.
Southern and Northern Blots
In southern blotting probes of known sequence are hybridized to target DNA fragments that have
been separated by gel electrophoresis. The hybridization procedure in which DNA is the target
sequence in the gel, and RNA or DNA is the probe, is called Southern blot. The DNA is first denatured
to single strands and then transferred into a synthetic membrane. Denaturant prevent secondary
structures. Then the membrane is exposed to a labelled probe. If the probe is complimentary to a
fragments, it binds and can be located.
A Northern uses RNA as the target sequence and DNA or RNA as the probe to detect gene
expression. RNA molecules are separated and not DNA molecules. Used to identify mRNA from
specific genes.
Other hybridization methods
Replica plating = cells on filter are lysed and release their DNA, DNA becomes single stranded and
fixed on the filter, than exposed to labelled probe to allow hybridization and wash the rest away,
11.1 restriction enzymes and nucleic acid separation
In vivo = in living organisms
In vitro = in test tube.
Restriction enzymes recognise specific sequences and cut them, protect cell from other DNA. Can’t
cut own DNA because on every sequences in its own DNA hangs a methyl-group that protects the
DNA.
Mechanisms of restriction enzymes
3 classes: I, II and III. I and III bind on sequences on DNA but cut a few sequences later. II cut exactly
at the sequence.
A sequence: 4-8 bp, written from right to left and left to right the same -> palindromes.
Sticky(forms two single strand DNA ends) and blunt ends(right in half)
Modification: protection form restriction
Modification enzymes protect own DNA against restriction enzymes by adding a methyl-group at the
sequences. This modifying is called methylases.
Gel electrophoresis: Separation of DNA Molecules
Based on moleculesize. Also used to make sure that the DNA amplification was successful. Separating
charged molecules by an electrical field. Gel made of agarose for separating DNA fragments. DNA
move from negative to positive pole because DNA is negative, in this way the DNA gets separated. By
adding ethidium bromide the DNA will fluoresce under UV light.
11.2 Nucleic Acid Hybridization
Single stranded DNA can bind with other single stranded DNA or RNA , can be used in detecting,
characterizing and identifying segments of DNA. Known single stranded DNA segments that are used
are called nucleic acid probes, can be radioactive labelled.
Southern and Northern Blots
In southern blotting probes of known sequence are hybridized to target DNA fragments that have
been separated by gel electrophoresis. The hybridization procedure in which DNA is the target
sequence in the gel, and RNA or DNA is the probe, is called Southern blot. The DNA is first denatured
to single strands and then transferred into a synthetic membrane. Denaturant prevent secondary
structures. Then the membrane is exposed to a labelled probe. If the probe is complimentary to a
fragments, it binds and can be located.
A Northern uses RNA as the target sequence and DNA or RNA as the probe to detect gene
expression. RNA molecules are separated and not DNA molecules. Used to identify mRNA from
specific genes.
Other hybridization methods
Replica plating = cells on filter are lysed and release their DNA, DNA becomes single stranded and
fixed on the filter, than exposed to labelled probe to allow hybridization and wash the rest away,
