Genome projects and gene tech

Question 1

genome definition

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

Proteome definition

Answer 3

the full range of proteins produced by cells

Question 4

Recombinant DNA definition

Answer 4

a cell having two or more sources of DNA

Question 5

Features of genetic code

Answer 5

universal
degenerate
non-overlapping

Question 6

5 steps in recombinant DNA tech

Answer 6

1. isolation of genes
2. insertion
3. transformation
4. identification
5. growth

Question 7

how can you obtain fragments of DNA

Answer 7

-mRNA –> cDNA via reverse transcriptase
-restriction enzymes
-gene machine

Question 8

How can we use reverse transcriptase to create DNA fragments

Answer 8

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

Question 9

Advantages of using reverse transcriptase

Answer 9

-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 10

How can we use restriction endonucleases to create DNA fragments

Answer 10

-Restriction endonucleases hydrolyse DNA at specific base sequences
-These are usually either side of a desired gene
-These recognition sequences are often palindromic = base pair read is the same in opposite directions
-The DNA sample is incubated with the specific restriction endonucleases which hydrolyses the DNA into fragments wherever the recognition sequence appears
-If the target gene has recognition sequences before and after the target gene, the fragments will contain the desired gene

Question 11

What can we add to DNA fragments

Answer 11

promoter regions = allow transcription factors to bind, allowing gene to be expressed

terminator region = cause transcription of gene in interest to stop

Question 12

Gene machine process to create DNA fragments

Answer 12

-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

Question 13

Advantages of Gene machine

Answer 13

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

Question 14

Insertion of genes into a vector process

Answer 14

-Isolated Target DNA fragment inserted into the vector DNA by cutting open the vector DNA using the SAME restriction endonuclease that was used to isolate the DNA fragment
-produces complementary sticky ends between the ends of DNA fragment and cut ends of vector DNA
-Target DNA fragment anneals to vector DNA by complementary base pairing between their sticky ends
-DNA ligase is used to join the DNA fragment and vector DNA at the sugar phosphate backbone by forming phosphodiester bonds.
-The combined DNA fragment and vector DNA is recombinant DNA

Question 15

Vector definition

Answer 15

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

Question 16

Transformation producing recombinant organisms process

Answer 16

1. make artificial DNA with correct sequence of bases
2. using DNA polymerase
3. cut plasmid open
4. with same restriction endonuclease
5. sticky ends attached;
6. use DNA ligase to join
7. return plasmid to bacterial cells

Question 17

Why must transformed cells be identified

Answer 17

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

Question 18

Cloning via bacteria

Answer 18

-Once you have identified the transformed bacterial cells then you can culture these bacteria as they reproduce by binary fission to produce genetically identical cells with the desired gene
-These organisms will produce large quantities of the desired protein

Question 19

cloning via PCR

Answer 19

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 cycle many times

Question 20

Why is PCR more appropriate than cloning via bacteria

Answer 20

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

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 evade 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 releases 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 gen
-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 germline therapy

Answer 25

-imperfect due t random fusion of gametes
-denial of human rights
-used to eliminate disease but also enhance favourable characteristics

Question 26

limitations of somatic gene therapy

Answer 26

-not all cells take up new DNA
-not all cells express DNA allele
-body can produce an immune response to the vector

Question 27

Processing of genetic screening using DNA probes and DNA hybridisation

Answer 27

1. sequence target gene
2. create DNA fragments of complementary target gene
3. conduct PCR to obtain large sample of DNA fragments
4. make probe = DNA fragments + marker
5. target DNA strand separated by heating then cooled with probe
6. probe binds to target ssDNA
7. washed to remove excess probe
8. hybridised DNA observed through special microscope

Question 28

what is DNA probe

Answer 28

-A short, single stranded DNA molecule with a complementary base sequence to DNA fragment to be located which is radioactive or labelled by a fluorescent molecule

Question 29

What does VNTR mean?

Answer 29

Variable Number Tandem Repeats

Question 30

Process of DNA fingerprinting

Answer 30

1. DNA extracted from sample
2. DNA hydrolysed into segments by restriction endonucleases
3. VNTR’s left intact
4. DNA fragments separated using electrophoresis
5. Mixture put into wells on gel and electric current passed through
6. Immerse gel in alkaline solution to separate DNA strands
7. Southern blotting=cover with nylon to absorb DNA
8. DNA fixed to nylon membrane by UV light
9. Radioactive marker with probe added which hybridises to target fragments
10. areas with probe bidentified using X-ray film