DNA Technologies: 1 Flashcards
Certaines endonucléases dites ________________________ ou
enzymes de restriction coupent l’ADN bicaténaire à des endroits
caractérisés par des séquence de nucléotides spécifiques,
dites ________________________.
endonucléases de restriction
Sites de Restrictions
Pourquoi les bactéries ont
des enzymes de restriction?
Servent à la défense contre les virus des bactéries
(les bactériophages) en coupant l’ADN étranger en morceaux
Les enzymes de restriction font partie d’un système de défense de bactéries
Ce sont des enzymes qui reconnaissent et coupent l’ADN à des
séquences spécifiques
What is special about enzymes de restriction?
majorité reconnaissent de séquences palindromiques
Comment séparer et identifier les fragments d’ADN coupés après digestion avec des enzymes de restriction?
Une fois coupés, les fragments d’ADN peuvent être séparés
selon la taille par Électrophorèse sur Gel d’Agarose
Explain how Électrophorèse sur Gel d’Agarose works?
DNA is acidic → charged negatively
Given a current in the media, the DNA will try to go towards the positive node
The agarose serves as a mesh, where smaller DNA can travel faster
We can use it to measure DNA segments
Molécule fluorescente qui s’intercale à l’intérieur de la double hélice, a way to detect DNA?
Bromure d’Ethidium
What is the recombinant DNA technique? What does it require?
Use DNA Ligase to join two DNA fragments that were created using restriction enzymes
Needs ATP
DNA Ligase used in bouts francs vs bouts collant? What are these?
bouts francs: blunt end, straight cut
bouts collant: sticking out ends, stronger interaction
L’efficacité de ligation de bouts francs est 100 fois plus faible que celle de la ligation de bouts collants
What is a plasmid?
cercle d’ADN bicaténaire qui peut se répliquer de façon autonome dans un organisme hôte
Just like DNA but we can have multiple copies
Can insert a DNA in the plasmid and then the plasmid will be incorporated in the Bacterial DNA
How can you introduce a new protein gene in a bacteria?
Using plasmids:
Using Ligases and bouts franc we can introduce any DNA sequence into plasmids → vector
vecteur est un plasmide, qui a été manipulé en laboratoire, dans lequel on peut insérer (cloner) des fragments d’ADN
We can add antibiotic resistance genes along with our target DNA. As such, we can select for only the bacteria that have indeed incorporated the plasmid by putting the bacteria in a antibiotic environment and selecting for the ones that survive
What is dénaturation thermique?
Melting Temperature: Tm
Using a double brin DNA and putting in high temperature condition which will lead to the partial denaturing and thus 2 simple strands that can be used by DNA Polymerase (5’ → 3’)
What is HYBRIDATION?
L’ADN dénaturé (simple brin) peut se renaturer (re-hybrider) en ADN
double brin, lorsqu’on revient aux bonnes conditions (initiales)
Renaturation= Hybridation
Renaturation selon la règle G-C et A-T
Explain sanger’s sequencing method?
technique for determining the sequence of nucleotide bases in a piece of DNA
In this method, ddNTPs are used (in low concentration; 4 tubes) to terminate the synthesis reaction. Compared to dNTPs, ddNTPs have an oxygen atom removed from the ribonucleotide, hence cannot form a link with the next nucleotide
Gel Electrophoresis: The denatured fragments are separated by gel electrophoresis and the sequence is determined.
https://youtu.be/KTstRrDTmWI
Comment on produit beaucoup d’ADN à partir
de quelques molécules?
Polymerase Chain Reaction
Explain Polymerase Chain Reaction?
involves using short synthetic DNA fragments called primers to select a segment of the genome to be amplified, and then multiple rounds of DNA synthesis to amplify that segment.
1) Séparation des brins (95C)
2) Hybridation des amorces (55C)
3) Synthèse par la ADN polymérase
What is TaqI ADN Polymerase?
Instead of having to introduce DNA polymerase everytime the tube is heated to 95*, Taql polymerase comes from some organism that lives in very high temperatures
thus, taql pol can survive heat shock and so you do not have to resuply the sample with polymerase
SÉQUENCES RÉPÉTÉES EN TANDEM and PCR?
PCR to multiply DNA
The sequences around the locus of the STRs are conserved between individuals
Because STR regions are very variable from one individual to another, we can isolate them
They vary based on their number of repetition
So now we can cut all of the STRs of one individual and based on the lengths of them we can identify their identity using PCR
What are SNPs? Clinical relevance?
Single nucleotide polymorphisms
Variations that occur during replication
They are very different between two individuals
They exist in coding and non coding DNA
Clinical:
They could appear on site de restriction, makes one medication less or more efficient for an affected individual
Des fois, ces différences sont suffisantes pour qu’une personne soit en santé ou très malade
déterminer les probabilités d’avoir un cancer or pas
SNP vs mutation?
A mutation occurs in less than 1% of a populattion an SNP occurs in more than 1%
Describe CRISPR-Cas 9?
Enzyme based Systeme de defense bacterium against phages
Recognize the DNA that is virus DNA and put it in a specific region of the DNA.
They can then create small parts of the virus DNA and express it in their cytoplasm.
These bacteria will then be resistant to this phage because it will be able to recognize the virus DNA and be able to destroy it
CRISPR-Cas 9 and clinical relation?
We can use this powerful enzyme to target a specific gene sequence in the human genome and perform gene editing
The cells really really want to fix the separated strands
They can either put them together, or we can introduce a prefered sequence to add between the two. I.e., add a new sequence to the human DNA
Although this is currently likely to lead in mutations as the sequence could potentially bind at many parts of the DNA
Sur un gel d’agarose, vers quel pôle migrent les fragments d’ADN?
pôle positif du gel. Cela s’explique par le fait que l’ADN est acide et donc chargé négativement
