Medical Genomics – Lecture 3 – Molecular cloning and genome
sequencing
Cloning by plasmids
Clone; collection of completely identical items
Animal cloning
Molecular/DNA cloning; use because you need more than 1 copy of a DNA
molecule to do many things. For example, sequence analysis,
genetic engineering etc.
Vector; general name for all thins in where you can clone
Plasmid; smaller pieces of DNA. They have origin of replication.
Viruses; may harbor larger pieces of DNA
Yeast artificial chromosomes (YACs); grow slower than bacteria
but they have chromosomes so you can add chromosome extra.
Very large pieces of DNA (2000 kb)
Bacterial artificial chromosomes (BACs); smaller pieces of DNA
(300 kb)
How to clone a gene? --> ampflication (versterking)
1. Need DNA to clone
2. Restrict (knippen) the DNA to sticky ends
3. Insert the DNA into the plasmid by DNA ligase (plakken)
4. Cloning starts.
Manipulating DNA
Restriction/modification is discovered by bacteria as protection against foreign DNA.
First; Bacteriophage attack the bacteria, cell lysis.
Later; Bacteriophage attack the bacteria, bacteria restrict the bacteriophage DNA cell
will be alive.
But, how does the bacteria protect its own DNA against restriction enzyme? By
modification of the restriction sites by methylation.
Palindrome
5’ AGCT 3’
3’ TCGA 5’
It is exactly the opposite.
Restriction enzymes recognize the palindromic sequences.
Restriction enzymes for cloning
The DNA strands are restricted (knippen). The 2 sticky ends
are become together by hydrogen bonding. DNA ligase will
covalently link the DNA backbones.
Analyze the length of sequence
By gel-electrophoresis. - on the top + on the end.
Library
sequencing
Cloning by plasmids
Clone; collection of completely identical items
Animal cloning
Molecular/DNA cloning; use because you need more than 1 copy of a DNA
molecule to do many things. For example, sequence analysis,
genetic engineering etc.
Vector; general name for all thins in where you can clone
Plasmid; smaller pieces of DNA. They have origin of replication.
Viruses; may harbor larger pieces of DNA
Yeast artificial chromosomes (YACs); grow slower than bacteria
but they have chromosomes so you can add chromosome extra.
Very large pieces of DNA (2000 kb)
Bacterial artificial chromosomes (BACs); smaller pieces of DNA
(300 kb)
How to clone a gene? --> ampflication (versterking)
1. Need DNA to clone
2. Restrict (knippen) the DNA to sticky ends
3. Insert the DNA into the plasmid by DNA ligase (plakken)
4. Cloning starts.
Manipulating DNA
Restriction/modification is discovered by bacteria as protection against foreign DNA.
First; Bacteriophage attack the bacteria, cell lysis.
Later; Bacteriophage attack the bacteria, bacteria restrict the bacteriophage DNA cell
will be alive.
But, how does the bacteria protect its own DNA against restriction enzyme? By
modification of the restriction sites by methylation.
Palindrome
5’ AGCT 3’
3’ TCGA 5’
It is exactly the opposite.
Restriction enzymes recognize the palindromic sequences.
Restriction enzymes for cloning
The DNA strands are restricted (knippen). The 2 sticky ends
are become together by hydrogen bonding. DNA ligase will
covalently link the DNA backbones.
Analyze the length of sequence
By gel-electrophoresis. - on the top + on the end.
Library
