B21 - Recombinant DNA technology

Question 1

What does recombinant DNA technology allow?

Answer 1

Genes to be manipulated, altered, and transferred from one organism to another

Question 2

Define recombinant DNA

Answer 2

DNA of 2 different organisms combined by cloning, resulting in transgenic or genetically modified organism

Question 3

What are the steps in making proteins using DNA technology?

Answer 3

1. Isolation of DNA fragments of interest (gene)
2. Insertion of DNA fragment into a vector
3. Transformation or transfer of DNA into host cells
4. Identification of transformed cells using gene markers
5. Growth/cloning of host cells

Question 4

What is the purpose of restriction endonucleases and how do they work?

Answer 4

Bacterial enzymes that hydrolyse viral DNA
They cut a DNA double strand at a specific sequence - recognition site

Question 5

What are the two types of DNA ends created by restriction endonucleases?

Answer 5

Blunt end
Sticky end

Question 6

What does a sticky end refer to?

Answer 6

A staggered cut across the recognition site, creating short single-stranded overhangs

Question 7

What is the role of DNA ligase?

Answer 7

Reforms phosphodiester bonds to join DNA fragments together

Question 8

What is a gene machine used for?

Answer 8

To synthesize genes by designing overlapping oligonucleotide fragments

Question 9

What is the function of reverse transcriptase in cDNA synthesis?

Answer 9

To make complementary DNA (cDNA) from mRNA

Question 10

What is the purpose of PCR?

Answer 10

Automated method of copying DNA fragments

Question 11

What are the components required in a PCR mix?

Answer 11

DNA template
DNA polymerase
DNA nucleotides (dNTP)
ATP
Primer

Question 12

What is gel electrophoresis used for?

Answer 12

To separate DNA fragments based on their size

Question 13

What charge do all DNA molecules have?

Answer 13

Negative charge, because of the presence of phosphate groups in nucleotides

Question 14

What is a DNA probe and why is it used?

Answer 14

Short ssDNA with a label attached that binds to specific DNA sequences
Can locate specific alleles

Question 15

What is the purpose of genetic screening?

Answer 15

Especially important, when a disease is caused by a recessive allele, so carrier parents do not show signs of it

Question 16

What is the benefit of personalized medicine?

Answer 16

Allows more effective drug prescription based on the patient’s genetic profile, for example a lot of drugs need to be activated by specific enzymes in the patient’s body. If we know a patient lacks such enzyme, other drug can be prescribed.

Question 17

What are variable number tandem repeats (VNTR)?

Answer 17

Non-coding sequences in DNA that are unique for every individual. The closer related are two individuals, the more similar the fragment pattern is

Question 18

What is in vivo cloning?

Answer 18

Cloning that occurs in a living organism

Question 19

What role do plasmids play in gene cloning?

Answer 19

They serve as vectors to carry target genes into host cells

Question 20

What is the purpose of transformation in bacteria?

Answer 20

To introduce plasmids containing target genes into bacterial cells

Question 21

How can successfully transformed bacteria be identified?

Answer 21

Antibiotic resistance selection
Marker genes
Replica plating

Question 22

What is the significance of the green fluorescent protein (GFP) gene in bacterial transformation?

Answer 22

It helps identify colonies that have successfully taken up the plasmid with the target gene

Question 23

What are the advantages of in vivo cloning?

Answer 23

Delivery of gene into another organism for gene therapy
No risk of contamination
Very accurate (more than PCR)
Cuts out specific genes
Large amounts of gene product can be made

Question 24

What are the steps in the conversion of mRNA to cDNA?

Answer 24

1. This mRNA is extracted from these cells and purified
2. Reverse transcriptase is used to make complementary DNA (cDNA) of an mRNA molecule forming an mRNA-cDNA hybrid
3. The mRNA is hydrolysed off the cDNA
4. Another complementary DNA strand is synthesised by DNA polymerase using the cDNA as a template

Question 25

Why can mRNA be preferred to DNA when isolating fragments?

Answer 25

Specialised cells contain a high concentration of certain mRNA molecules in their cytoplasm

Question 26

What are the advantages to the gene machine?

Answer 26

- Whole process is computerised so it is fast and accurate. - Gene can be sequenced to ensure there are no errors. - Gene does not contain introns or other non-coding DNA, so prokaryotic cells can be used for transcription and translation

Question 27

What are the steps of PCR (Polymerase chain reaction)?

Answer 27

1. Denaturing the double stranded DNA. At 95 degrees the H-bonds break and the 2 DNA strands separate, forming 2 single stranded DNA fragments. 2. The mixture is cooled to 55 degrees and the primers join (anneal) to the complementary ends of the ssDNA fragments. 3. Synthesis of new DNA. Temperature is raised to 72 and the DNA polymerase, starting at the primers, join complementary nucleotides to form a new strand of DNA. Now we have 2 new dsDNA fragments There becomes an exponential growth in the number of copies produced. 2^n with n being the number of cycles

Question 28

How can antibiotics be used to identify successfully transformed bacteria?

Answer 28

If the plasmid had antibiotic resistance gene on it, expose bacteria to the antibiotics. Those that took up plasmid will survive, the others will die.