Technologies

Question 1

Define Recombinant DNA Technology.

Answer 1

Involves using enzymes and various laboratory techniques to manipulate and isolate DNA segments of interest.

Question 2

List the key innovations in genetic technologies.

Answer 2

PCR; allows small quantities of specific DNA to be amplified.
Nuclei acid hybridization; techniques allowing visualization of specific DNA sequences.
DNA sequencing; development of quick and accurate sequencing.
CRISPR-Cas systems; to accurately edit of genome sequences.

Question 3

What is amplification and how it may be done?

Answer 3

Amplification; use DNA polymerase to create multiple copies.
This can be done with DNA cloning or PCR.

Question 4

Define Vector Molecule(DNA Cloning).

Answer 4

Vector molecules are DNA molecules that is used as a vehicle (plasmid) to carry a particular DNA segment into a host cell as part of DNA cloning.

Question 5

Define restriction endonucleases (RE).

Answer 5

Are enzymes used to cut large DNA molecules into smaller pierces and vectors. These cut pieces of DNA can then be joined to vector molecules by DNA ligase to produce recombinant DNA.

Question 6

REs are produced by..?

Answer 6

REs are produced by bacteria and used for defense against viruses. (REs make cuts in DNA at specific nucleotide sequences it recognizes, bacteria protects itself by modifying this recognition sequence)

Question 7

Define engineered nucleuses (ENs) and what they consist of.

Answer 7

ENs are designed to cut DNA at any predetermined DNA sequence. It consist of part of an RE + another protein that recognizes and binds to a specific DNA sequence (e.g. Zinc finger nucleuses; use a DNA binding protein called zinc finger).

Question 8

Define CRISPR-Cas.

Answer 8

It’s a DNA cutting precision tool which was first seen in bacteria, protecting themselves from forgiven genetic elements(e.g. plasmids).

Question 9

Generally, how are DNA fragments located?

Answer 9

This is done via isolating DNA with RE and separates fragments on gel.

Question 10

Define and outline the process of southern blotting.

Answer 10

Denature DNA & transfer to solid medium. (Watch video for more info)

Question 11

Define northern blotting.

Answer 11

RNA is transferred from gel to a membrane. A probe is used to reveal size of mRNA molecule, its abundance, etc…

Question 12

Define western blotting.

Answer 12

Transfer of protein from gel to a membrane. Probe used to reveal size of a protein and pattern of expression.

Question 13

What event occurs creating RFLPs?

Answer 13

SNPs sometimes affect recognition site for REs, creating restriction fragment length polymorphisms.

Question 14

How did RLFPs impact genetics? How may they be used in clinical application?

Answer 14

RFLPs were the first widely used DNA markers, they were used in genetic linkage map, and was used along with REs to show that in some families with genetic disorder, certain polymorphisms were inherited together with the disease state.

Question 15

What are linkage analysis and gene tracking used for?

Answer 15

Both were used for carrier detection, antenatal diagnosis, etc testing for a wide range of genetic disorders. This is a crucial step leading later to the location and identification of genes involved.

Question 16

Define cloning vectors. List its key properties and give an example.

Answer 16

DNA molecules that accept DNA fragments & replicate them when introduced into the host cells(e.g. E. Coli plasmids).
Key properties include: containing restriction sites to allow in detection of DNA fragments to be cloned, and capable of replication in host cells for independent replication of vector DNA.

Question 17

Define transgenic organisms.

Answer 17

Cloned gene inserted into organisms are called transgenic organisms or GMOs.

Question 18

Define and outline the process of chromosome FISH, what might this technique be used for? (REVIEW)

Answer 18

FISH is a cytogenetic technique which uses fluorescent DNA probes to target specific chromosomal location with the nuclear system.

Question 19

What are the two essential enzymes used to make recombinant DNA?

Answer 19

1. Restriction enzymes used to cut DNA at specific recognition sites.
2. DNA ligase forms phosphodiser bonds to join a restriction fragment and vector DNA.

Question 20

Define expression vectors and how they differ from cloning vectors.

Answer 20

Expression vectors are designed to ensure mRNA expression of a cloned gene(unlike vectors described previously).

Question 21

List 3 recombinant proteins that are medicinally used.

Answer 21

Recombinant factor VIII & IX; used to treat different forms of hemophilia.
Insulin; treats some forms of diabetes
Tissue plasminogen activator; use in life threatening emergencies

Question 22

What are the advantages and disadvantages of DNA cloning?

Answer 22

Adv; made genome projects possible. Was a corner stone in genetic innovation and biotech which lead to research on gene structure, analysis of expression, and gene function.
Dis; time consuming, laborious, not suited to doin rapid parallel amplifications of the same DNA sequence.

Question 23

Define NAH and Outline the process of nuclei acid hybridization.

Answer 23

NAH is used to track DNA and RNA, it relies on the extent or specificity of base pairing.
1. Test nucleic acid population with sequence of interest is first made single stranded (denatured).
2. Mixed with a probe population of known denatured nucleic acids and allowed to renature forming an artificial duplex.
3. Object is to identify stable heteroduplexes where a single stranded sequence of interest in the sample has hybridised with a known sequence within the probe population.

Question 24

Outline microarray hybridization.

Answer 24

In microarray hybridisation, many thousands of unlabelled oligonucleotide probe populations are attached to a solid surface in a regular grid pattern. (Watch video)

Question 25

Define molecular cytogenetics.

Answer 25

Scientific discipline concerned with the structure and function of genes at the molecular level.
Combination of conventional cytogenetics and molecular genetic technology.

Question 26

Outline the process of generating cDNA.

Answer 26

(RNA cannot be cloned but DNA copies can be made!)

• A reverse transcriptase copies RNA template to make a
  complementary DNA (cDNA) strand.
• Original RNA then destroyed with a ribonuclease & copied DNA is then itself copied to produce double-stranded cDNA.
• Total ds cDNA from cells can be used to make cDNA library.