Recombinant DNA technology (complete)

Question 1

Define the Polymerase Chain reaction

Answer 1

Method to replicate DNA in the lab. Allows for amplification of genes before they are reinserted, making recombinant DNA.

Question 2

What are the requirements for the polymerase chain reaction?

Answer 2

• Free nucleotides
• Primers
• DNA Polymerase
• Thermocycler
• The DNA fragment to be copied.

Question 3

What are primers (PCR)?

Answer 3

• Short sequences of nucleotides that are complementary to DNA fragments.
  They prevent the DNA strands from rejoining and provide a starting sequence for DNA replication in the PCR.

Question 4

Describe the process of the Polymerase chain reaction.

Answer 4

• The DNA is heated to 95 degrees to break the hydrogen bonds between the bases and separate the DNA strands.
• The single strands are mixed with complementary primers to prevent them rejoining and the mixture is cooled to 55 degrees to allow the primers to bind.
• Then free nucleotides and DNA polymerase are added and the mixture is heated to 70 degrees, which is the optimum temp for DNA polymerase, so transcription can occur. The DNA polymerase forms new phosphodiester bonds between the free nucleotides, and two new double stranded DNA are formed.

Question 5

Name the five steps in producing a protein using DNA technology.

Answer 5

• Isolation
• Insertion
• Transformation
• Identification
• Growth/cloning

Question 6

Name the different ways of forming DNA fragments.

Answer 6

• Using reverse transcriptase
• Using restriction endonucleases
• Using the gene machine.

Question 7

Describe how DNA fragments are formed using Reverse transcriptase.

Answer 7

• Take the desired mRNA that codes for the desired product (e.g: insulin)
• This acts as a template on which a single stranded complementary copy of DNA (cDNA) is formed using reverse transcriptase, forming a double stranded mRNA.
• This formed cDNA is isolated by hydrolysis of the double stranded mRNA using an enzyme.
• A new strand of DNA is formed using the isolated cDNA as a template strand and DNA polymerase to join the complementary nucleotides, forming a double stranded cDNA as a copy of the desired gene.

Question 8

Describe how DNA fragments are formed using restriction endonucleases.

Answer 8

• The restriction endonucleases cut the desired gene at a specific recognition site, producing either sticky ends or blunt ends on the DNA fragment.

Question 9

Describe how DNA fragments are formed using the Gene Machine.

Answer 9

• The desired sequence is established from the protein, which is coded into the computer and checked for biosafety (the right protein is being made).
• Nucleotides are combined (automated = one at a time), forming oligonucleotides.
• These are then joined together to make a single strand.
• DNA polymerase is used to make a double strand (PCR) with sticky ends.

Question 10

How is a DNA fragment prepared to be added into a vector?

Answer 10

• A promoter region must be added to allow for RNA polymerase and transcription factors to bind.
• A terminator region must be added to stop the transcription at the end of the desired gene.

Question 11

Describe the insertion of a gene (using restriction endonucleases) into a vector plasmid.

Answer 11

• The sticky ends on the gene and plasmid vector must be cut by the same restriction endonucleases to ensure they are complementary.
• The gene is then inserted into the plasmid, using DNA ligase to bond the sugar-phosphate backbones to insert the gene.

Question 12

How are the recombinant plasmids introduced into host cells? (Transformation)

Answer 12

• Mix the plasmids with the host cells in a medium contains calcium ions.
• The calcium ions make the bacterial (host) cells permeable, allowing the plasmids to freely pass through into the cell.

Question 13

Name the three ways of identifying successful host cells in taking up recombinant DNA, using gene markers.

Answer 13

• Using antibiotic markers
• Using fluorescent markers
• Using enzymes

Question 14

Describe how to identify successful host cells using antibiotic resistant markers.

Answer 14

• The plasmid contains two genes providing two seperate resistances to different antibiotics.
• Insert out desired gene into one of the pre-existing resistant genes, causing the gene not to work so the plasmid is no longer resistant to that antibiotic.
• Let the bacteria grow on agar treated with the (still resistant to) antibiotic.
• Make a replica plate of those colonies grown onto a different agar plate treated with the (hopefully) not resistant antibiotic.
• Any missing colonies indicate cells which have taken up the plasmid as they are no longer resistant to the affected genes antibiotic.

Question 15

Describe how to identify successful host cells using Fluorescent markers.

Answer 15

• Insert the gene of interest into a gene for a fluorescent protein.
• Insert this complex into a plasma vector.
• Transfer the plasmids into host bacterial cells.
• Any cells that successfully take up the plasmid will be glowing on the petri dish.

Question 16

Describe how to identify successful host cells using Enzyme markers.

Answer 16

• Insert the gene of interest into a gene that codes for an enzyme, so deactivating the enzyme.
• There will be a particular substrate that changes colour when an enzyme acts upon it.
• Adding the vectors into bacterial cells and letting them grow on agar medium that contains the substrate.
• Any colonies that didn’t change colour have taken up the plasmid.

Question 17

Define Genetic screening.

Answer 17

Screening of embryos to check for mutant alleles.
Can determine the probability of couples having offspring with a genetic disorder.

Question 18

Describe the process of DNA hybridisation. (Genetic screening)

Answer 18

• The DNA is heated to break the hydrogen bonds between bases, forming two single strands of DNA.
• DNA probes (complementary to the mutated genes) are added and the DNA cooled to bind to complementary sequences of bases.
• Wash the mixture to remove any unbound DNA probes.
• The DNA is now identifiable by either radioactivity or fluorescence.

Question 19

What is Gel electrophoresis?

Answer 19

• Used to separate DNA fragments according to their size.

Question 20

How does gel electrophoresis work?

Answer 20

• The DNA fragments are added to agar gel and a voltage is applied across it.
• The resistance of the gel means the larger the fragments the slowest they move.

Question 21

Name the five stages in Genetic fingerprinting.

Answer 21

• Extraction
• Digestion
• Separation
• Hybridisation
• Development

Question 22

Describe the process of genetic fingerprinting.

Answer 22

• DNA is extracted from the sample by separating it from the rest of cell.
• The DNA is cut into fragments using the same restriction endonucleases to cut close to the target DNA.
• The fragments are separated using gel electrophoresis, which is immersed in alkali to separate the double strands into single strands. The DNA fragments are then transferred from gel to nylon membrane.
• Radioactive or fluorescent DNA probes are used to bind with VNTRs. The probes have base sequences which are complementary to the VNTRs base sequences, and bind to them under specific conditions.
• X-ray film is put over the nylon membrane, and is exposed to radiation from the radioactive probes, and an X-ray film is developed to reveal dark bands where the radioactive DNA probes have attached.

Question 23

Name some uses of genetic fingerprinting.

Answer 23

• Identifying criminals
• Paternity testing
• Testing genetic variability in a population.
• Selective breeding of plants and animals.
• Identifying microbes.
• Medical diagnosis of a disease.