DNA Technologies: 1

Question 1

Certaines endonucléases dites ________________________ ou
enzymes de restriction coupent l’ADN bicaténaire à des endroits
caractérisés par des séquence de nucléotides spécifiques,
dites ________________________.

Answer 1

endonucléases de restriction
Sites de Restrictions

Question 2

Pourquoi les bactéries ont
des enzymes de restriction?

Answer 2

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

Question 3

What is special about enzymes de restriction?

Answer 3

majorité reconnaissent de séquences palindromiques

Question 4

Comment séparer et identifier les fragments d’ADN coupés après digestion avec des enzymes de restriction?

Answer 4

Une fois coupés, les fragments d’ADN peuvent être séparés
selon la taille par Électrophorèse sur Gel d’Agarose

Question 5

Explain how Électrophorèse sur Gel d’Agarose works?

Answer 5

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

Question 6

Molécule fluorescente qui s’intercale à l’intérieur de la double hélice, a way to detect DNA?

Answer 6

Bromure d’Ethidium

Question 7

What is the recombinant DNA technique? What does it require?

Answer 7

Use DNA Ligase to join two DNA fragments that were created using restriction enzymes

Needs ATP

Question 8

DNA Ligase used in bouts francs vs bouts collant? What are these?

Answer 8

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

Question 9

What is a plasmid?

Answer 9

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

Question 10

How can you introduce a new protein gene in a bacteria?

Answer 10

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

Question 11

What is dénaturation thermique?

Answer 11

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’)

Question 12

What is HYBRIDATION?

Answer 12

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

Question 13

Explain sanger’s sequencing method?

Answer 13

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

Question 14

Comment on produit beaucoup d’ADN à partir
de quelques molécules?

Answer 14

Polymerase Chain Reaction

Question 15

Explain Polymerase Chain Reaction?

Answer 15

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

Question 16

What is TaqI ADN Polymerase?

Answer 16

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

Question 17

SÉQUENCES RÉPÉTÉES EN TANDEM and PCR?

Answer 17

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

Question 18

What are SNPs? Clinical relevance?

Answer 18

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

Question 19

SNP vs mutation?

Answer 19

A mutation occurs in less than 1% of a populattion an SNP occurs in more than 1%

Question 20

Describe CRISPR-Cas 9?

Answer 20

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

Question 21

CRISPR-Cas 9 and clinical relation?

Answer 21

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

Question 22

Sur un gel d’agarose, vers quel pôle migrent les fragments d’ADN?

Answer 22

pôle positif du gel. Cela s’explique par le fait que l’ADN est acide et donc chargé négativement