13.8 Genome Projects and Gene Technology

Question 1

Define genome

Answer 1

Complete set of genes in a cell

Question 2

What does ‘genome sequencing’ mean?

Answer 2

To know the exact sequence of bases that make up the entire DNA of an organism

Question 3

Define proteome

Answer 3

The full range of proteins produced by cells

Question 4

Define recombinant DNA

Answer 4

Refers to a cell having two or more sources of DNA

Question 5

5 steps of recombinant DNA technology

Answer 5

1. Isolation of genes
2. Insertion
3. Transformation
4. Identification
5. Growth/cloning

Question 6

How can fragments of DNA be produced?

Answer 6

• Conversion of mRNA to cDNA using reverse transcriptase
• Using restriction enzymes to cut a fragment
• Creating gene in ‘gene machine’

Question 7

How can we use reverse transcriptase to create DNA fragments?

Answer 7

-Free DNA nucleotides bind to single-stranded mRNA template via complementary base pairing
-Reverse transcriptase joins DNA nucleotides together to form single-stranded cDNA molecules
-DNA polymerase required to make cDNA double stranded

Question 8

Advantages of using reverse transcriptase

Answer 8

-mRNA is much easier to obtain
-Bacterial DNA doesn’t contain introns as bacteria don’t have enzymes needed for splicing
-To copy a gene which codes for a certain protein, mRNA can be isolated from the cytoplasm of cell types which produce the protein in large amounts

Question 9

How can we use restriction endonucleases to create DNA fragments?

Answer 9

1. Restriction endonucleases hydrolyse DNA at specific base sequences usually either side of a desired gene
2. Recognition sequences are often palindromic
3. DNA sample is incubated with the specific restriction endonucleases which hydrolyses the DNA into fragments wherever the recognition sequence appears
4. If the target gene has recognition sequences before and after the target gene, the fragments will contain the desired gene
   - Produces either blunt or sticky ends

Question 10

How can we use the gene machine process to create DNA fragments?

Answer 10

-Desired nucleotide sequence fed into a computer
-Synthesis of oligonucleotides (short sequences of nucleotides)
-Assembly of gene = oligonucleotides are overlapped then joined together and made double stranded using PCR
-Gene is inserted into a bacterial plasmid
FASTER THAN ENZYME CONTROLLED REACTIONS

Question 11

Advantages of the gene machine

Answer 11

DNA without introns
Artificial genes are easily transcribed and translated by prokaryotes, as they have no introns in their DNA

Question 12

Define vector

Answer 12

A vector is a DNA carrier eg. virus used to transfer foreign DNA into cells

Question 13

What is the process of insertion of genes into a vector?

Answer 13

1. Cut the vector DNA using the SAME restriction endonuclease that was used to isolate the DNA fragment.
2. Produces complementary sticky ends between the ends of DNA fragments and cut ends of vector DNA
3. Target DNA fragment anneals to vector DNA by complementary base pairing between their sticky ends
4. DNA ligase is used to join the DNA fragment and vector DNA by forming phosphodiester bonds via condensation reactions.
5. Combined DNA fragment and vector DNA= recombinant DNA

Question 14

What is the process of transformation?

Answer 14

Where recombinant DNA vector is transferred into a host cell (bacteria)

Question 15

Why must transformed cells be identified?

Answer 15

1. Not all vectors take up target DNA to become recombinant
2. Not all host cells become transformed, by taking up recombinant vectors

Question 16

Explain cloning via bacteria

Answer 16

1. Once transformed bacterial cells have been identified, these can be cultured as they reproduce by binary fission and produce genetically identical cells with the desired ‘inserted’ gene.
2. These organisms will produce large amounts of desired protein.

Question 17

Describe the process of cloning via PCR

Answer 17

1. Heat DNA to 95 degrees to break weak hydrogen bonds
2. Add primers and add nucleotides
3. Cool to 55 degrees to allow the binding of nucleotides and primers
4. Add thermostable DNA (Taq) polymerase
5. Heat to 75 degrees
6. DNA polymerase joins nucleotides together
7. Repeat the cycle many times

Question 18

Why is PCR more appropriate than cloning via bacteria?

Answer 18

Quicker so can produce millions of copies of target DNA within hours

Question 19

Benefits of recombinant DNA technology

Answer 19

• Develop medical applications to produce a faulty/lack of protein.
• Develop agricultural applications.
• Better understanding of biological processes

Question 20

Concerns regarding recombinant DNA technology

Answer 20

• Inserting new genes can disrupt other genes
• Introducing herbicide resistance genes to crop plants could result in transfer to wild species when they interbreed, producing herbicide-resistance weeds.
• Tech may be concentrated in hands of large corps/wealthy people

Question 21

How can we use viruses as vectors in gene therapy?

Answer 21

• If foreign DNA fragments are introduced to viral genetic material, it will insert the foreign gene at the same time as its own genetic material
• Viral DNA is cut using the same restriction endonuclease and joined to foreign DNA using DNA ligase
• The virus then acts as a vector

However

• Viruses can cause an immune response and the formation of cytotoxic T cells and memory B cells
• The next time the virus is given, the secondary response is stimulated, and antibodies produced
• To combat this, vector viruses are further modified to avoid the immune response

Question 22

How can we use liposomes as vectors in gene therapy?-

Answer 22

Liposomes are lipid droplets which can cross the phospholipid bilayer and release target DNA into the cell

However

• The DNA does not move into the nucleus and so does integrate into the genome of stem cells lining the airways
• So new daughter cells will not have the functional gene.
• From this DNA, small amounts of mRNA can be produced

Question 23

What is somatic gene therapy?

Answer 23

DNA transfer to our normal body tissue

Question 24

What is germline gene therapy

Answer 24

DNA transfer to cells that produce eggs or sperm

Question 25

Limitations of somatic gene therapy

Answer 25

• Not all cells take up new DNA
• Not all cells express DNA allele
• Body can produce an immune response to the vector

Question 26

Limitations of germline therapy

Answer 26

• Imperfect due t random fusion of gametes
• Denial of human rights
• Used to eliminate disease but also enhance favourable characteristics

Question 27

What is a DNA probe?

Answer 27

• (Short) single strand of DNA;
• Bases complementary (with DNA/allele/gene);

Question 28

Explain the processing of genetic screening using DNA probes and DNA hybridisation

Answer 28

1. Determine the sequence of the target gene
2. Create DNA fragments of complementary target gene
3. Conduct PCR to obtain a large sample of DNA fragments
4. Make probe = DNA fragments + marker
5. Target DNA strand separated by heating then cooled with the probe
6. Probe binds to target ssDNA
7. Washed to remove excess probe
8. Hybridised DNA observed through a special microscope
   Used to locate specific alleles of genes

Question 29

What does VNTR mean?

Answer 29

Variable Number Tandem Repeats
Regions of DNA between genes

Question 30

Explain the process of DNA fingerprinting

Answer 30

1. DNA extracted from the sample
2. DNA hydrolysed into segments by restriction endonucleases producing blunt ends
3. VNTR’s left intact
4. DNA fragments are separated using gel electrophoresis by putting into wells and an electric current is passed through
5. Immerse gel in alkaline solution to separate DNA strands
6. Add radioactive gene probes complementary to VNTRs to allow positions of fragments be visualised as bands.
7. Identify base sequence of interest using X-ray film.

Question 31

Name three techniques used by scientists to compare DNA sequences.

Answer 31

• Polymerase Chain Reaction
• DNA fingerprinting
• Gel electrophoresis