8-4 Gene technologies

Question 1

What is recombinant DNA technology?

Answer 1

• Recombinant DNA technology involves many ways of manipulating DNA, these processes are all detailed below.

Question 2

How is reverse transcriptase used to make DNA?

Answer 2

• The enzyme reverse transcriptase is an enzyme that is found in only some viruses and bacteria and catalyses the formation of a double strand of DNA form a single strand on RNA.
• This allows us to make working versions of DNA that act as genes, by extracting mRNA from cells where that gene is being expressed.

Question 3

How are restriction endonucleases used to cut DNA fragments?

Answer 3

• Restriction endonucleases are enzymes, extracted from bacteria, that cut DNA at specific sequences, usually six base pairs in length.
• The most useful restriction endonucleases are those that make staggered cuts, as they leave sticky ends on the DNA.
• The diagram shows the difference between sticky ends and blunt ends.

Question 4

Why are sticky ends important?

Answer 4

• Sticky ends are important because if the same restriction endonuclease is used to cut two DNA fragments, then the ends will be complementary.
• This allows them to attach before stronger covalent bonds form.

Question 5

Why is in-vivo gene cloning needed?

Answer 5

• If a DNA fragment was placed in a cell it would be digested by enzymes and therefore a vector is used to insert DNA into cells.
• They are the plasmids from bacterial cells that naturally occur.

Question 6

What are the ways that isolated DNA fragments can be placed in plasmids?

Answer 6

• Plasmids and genes are cut with the same restriction enzyme to create complementary ends sticky ends, this means that the inserted DNA and vector are complementary and can be joined.
• The fragments are incubated with the plasmids, if a plasmid takes up the insert, base pairing takes place between the complementary ends which are then sealed with the use of DNA ligase which forms phosphodiester linkages.
• A recombinant DNA molecule is created.

Question 7

What are gene markers used for?

Answer 7

• To check whether the DNA has been taken up by the bacteria.

Question 8

What are the different types of gene markers?

Answer 8

• Antibiotic restraint genes.
• Fluorescent markers.
• Enzyme markers.

Question 9

What is DNA profiling?

Answer 9

• DNA profiling is a forensic technique used to identify individuals by characteristics of their DNA.
• Can also be used to determine genetic relationships between organisms.
• E.g. PCR.

Question 10

What is polymerase chain reaction (PCR) used for?

Answer 10

• To amplify DNA by making millions of copies of a given DNA sample.

Question 11

How does a PCR work?

Answer 11

1. A reaction mixture is set up by mixing the DNA sample, primers, free nucleotides and DNA polymerase which is the enzyme involved in creating new DNA strands.
2. The mixture is then heated to 95 degrees to break the hydrogen bonds and to separate the two strands.
3. The mixture is then cooled to a temperature between 50-65 degrees depending on the type of primers used, so that they can bind to the strands.
4. Temperature is increased to about 70 degrees as a this is the temperature DNA polymerase works at. The DNA polymerase is called Taq polymerase and is from bacteria that live in hot springs.
5. DNA polymerase creates a copy of the sample by complementary base pairing using the free nucleotides.
6. This cycle is repeated around 30 times and gives rise to an amount of DNA sufficient to create a DNA profile.

Question 12

What is in-vitro cloning?

Answer 12

• This gene cloning can be done with PCR
• This is fast, automated, and reliable once conditions are established.
• This does not require living cells and can have problems such as contamination and errors.

Question 13

What in-vivo cloning?

Answer 13

• Gene cloning that can be done using recombinant plasmids in bacteria.
• This is accurate and useful as the gene is placed in cells where it can be expressed.
• The disadvantage though is it is very time consuming and requires monitoring of cell growth.

Question 14

What are DNA probes?

Answer 14

• A DNA probe is a short, single stranded DNA molecule that is designed to complementary to a sequence to be detected.
• DNA probes are made in smaller quantities and then amplified using PCR.
• The DNA labelling of the fragments either uses radioactive isotopes or a fluorescent dye which glows under certain wavelengths of light.

Question 15

What can DNA probes used for?

Answer 15

• DNA probes can be used in order to detect heritable conditions of health risks.

Question 16

What is genetic fingerprinting?

Answer 16

• Genetic fingerprinting is a technique that can detect differences in peoples DNA.
• It uses variable number tandem repeats (VNTRs) which are short repeating sequences or bases.
• The probability of two individuals having identical VNTRs is extremely low therefore VNTR analysis can be used in genetic fingerprinting.

Question 17

What is gel electrophoresis?

Answer 17

• Gel electrophoresis is process used to separate the DNA fragments and proteins according to their size using an electric current.

Question 18

What can genetic fingerprinting be used for?

Answer 18

• Genetic fingerprinting can be used in the fields of forensic science, medical diagnosis as well as animal and plant breeding.
• It is carried out as follows as show in the diagram below.