Recombinant DNA technology

Question 1

Summary
Recombinant DNA technology

Applications in gene analysis

Applications in genome analysis

Answer 1

..

Question 2

Recombinant DNA (rDNA)
5 points

Answer 2

1. Is a DNA constructed in the laboratory from 2 (or more) natural or artificial DNA fragments.
2. Digestion with the appropriate
   restriction enzymes.
3. The purified fragments are ligated using a DNA ligase.
4. Different DNA fragments can be successively ligated to produce rDNA
5. rDNA is then cloned via insertion into a vector and amplification into
   a host cell

Question 3

Molecular cloning

5 points

Answer 3

1. DNA fragment to be cloned + cut vector (with antibiotic resistance gene)
   Ligation ->
2. Complex: vector/insert (DNA construct)
3. Transformation: intro of DNA complex to host cells
4. Selection of the transformed cells on a solid growth media containing the
   selection agent.
5. Bacterial colonies containing the plasmid vector and the insert.

(Each colony is a clone containing
a single insert DNA.)

Question 4

rDNA is used in basic science

Answer 4

1. gene analysis

2. Genome analysis

Question 5

rDNA is used in Biotechnology

3 points

Answer 5

1. Production of recombinant proteins
2. Production of Genetically Modified Organisms (GMO)
3. Molecular vaccines

Question 6

Gene isolation
Complementary DNA (cDNA) libraries

Answer 6

Collections of bacterial clones containing cDNA representing expressed genes in a given tissue at specific conditions
and corresponding to a specific physiological situation.

Only expressed genes are represented.

Question 7

Gene isolation

Genomic-DNA (gDNA) libraries

Answer 7

Collections of bacterial clones containing fragments of genomic DNA digested by a restriction enzyme.

The entire genome is represented: Coding sequences, Regulatory sequences, “Junk” DNA

Question 8

Making cDNA libraries:

Answer 8

1. RNA extraction and mRNA purification.
2. cDNA synthesis and production of double stranded DNA.
3. Ligation of the adapters and their digestion.
4. Ligation of cDNA into the vector.
5. Packaging of the rDNA (cDNA in the vector) into the phage capsule.
6. Plating the phage library into host bacteria.

Question 9

Making gDNA libraries:

Answer 9

1. gDNA extraction and purification
2. Digestion of gDNA and vector with a restriction enzyme.
3. Ligation of DNA fragments into the digested vector.
4. Packaging of the rDNA (gDNA fragments inserted in
   the vector) into the phage capsule.
5. Plating the phage library into host bacteria.

Question 10

Probes

3 points

Answer 10

1. Labelled RNA or DNA fragments with a known sequence used to detect
   a complementary sequence in a DNA or RNA population.
2. Homologous probes: from the same organism

Heterologous probes: from a different organism, used to detect
similar sequences in the studied organism.

3. The isolated clone is then sequenced and the sequence
   analysed.

Question 11

Transcriptome (total expressed genes in a tissue) analysis

Answer 11

1. Expressed Sequence Tags (ESTs): Random sequencing of thousands of clones from a cDNA library prepared from a given tissue at specific conditions.

Question 12

Genome Analysis (entire genome.)

Answer 12

1. Sequencing DNA libraries containing fragments representing the entire genome of a given organism.
2. The obtained sequences are made available to the Scientific community via accessible databases: NCBI: National Center for Biotechnological Information

Question 13

Mapping genomes

Answer 13

Assigning/locating a specific gene to a particular region of a
chromosome and determining the location and relative distances between genes on the chromosome.

Question 14

Physical map

Answer 14

The physical, DNA-base-pair distances from one gene to another.

Question 15

Genetic linkage map

Answer 15

Order of genes on a chromosome and the relative distances between these genes.

Question 16

Associating gene with function/phenotype

Monitoring change in gene expression

2 points

Answer 16

1. Single gene approach

Northern blotting.

Quantitative Reverse transcription-Polymerase Chain Reaction (QRT-PCR).

2. Global approach
   Checking the expression of thousands of genes at the same time.

Microarray technology

RNA sequencing

Question 17

Associating gene with function/phenotype

Gene silencing - Knock down technologies - Antisense RNA

3 points

Answer 17

1. Used to reduce the expression of a gene to check the impact on the physiology.
2. Antisense RNA are RNA sequences complementary to the target RNA, they hybridize to it and block its translation.
3. Antisense RNA are produced by transforming the target organism with
   a cDNA that is transcribed into antisense RNA.

Question 18

Associating gene with function/phenotype

Gene silencing - Knock down technologies - RNA interference using small RNA

3 points

Answer 18

1. Small RNA are natural or synthetic double stranded RNA molecules of 20-25 nucleotides complementary to the target RNA.
2. they hybridize to the target RNA and block its translation. They lead to its degradation by specific RNases.
3. Antisense RNA and small RNA allow to identify gene function.

Question 19

Associating gene with function/phenotype

Knock-out Technology

5 points

Answer 19

1. Knock-out are collections of insertionnal mutants produced by rDNA technology.
2. A vector is used to transfer randomly or specifically a tDNA
   (transfer DNA) to a location in the genome to alter a gene at that locus.
3. Each mutant has an alteration in a single gene.
4. By tracing the tDNA it is possible to find which gene was affected.
5. By identifying the change in the phenotype it is possible to link gene with the function.