8B Genome Projects and Gene Technologies

Question 1

What is the genome?

Answer 1

Entire set of DNA, including genes in an organism

Question 2

What did the 2003 human genome project do?

Answer 2

Mapped entire sequence of human genome

Question 3

How do you sequence an entire genome?

Answer 3

Chop it up into smaller pieces and put back into order because methods only work on fragments of DNA

Question 4

What does sequencing of simple organisms help identify?

Answer 4

Their proteins

Question 5

What is the proteome?

Answer 5

All proteins made by an organism

Question 6

Why is it easier to determine the proteome of simple organisms?

Answer 6

Have less non-coding DNA which makes it easier to find proteome from genome

Question 7

Why does bacteria and viruses being simple organisms helpful in medical research?

Answer 7

Identify protein antigens and develop vaccine easier as less non-coding DNA

Question 8

Why is it hard to translate the genome of complex organisms?

Answer 8

• Contain large sections of non-coding DNA

- Contain regulatory genes

Question 9

What do regulatory genes do?

Answer 9

Determine when the genes that code for specific protein should be switched on or off

Question 10

How have sequencing methods changed over time?

Answer 10

• Less expensive
• Large scale
• More automated
• Faster

Question 11

What does recombinant DNA technology do?

Answer 11

Transfers a fragment of DNA from one organism to another

Question 12

What are transgenic organisms?

Answer 12

Organisms that contain transferred DNA

Question 13

Why can transferred DNA be used to produce a protein in cells of a recipient organism?

Answer 13

• DNA is universal

- Transcription and translation mechanisms are similar

Question 14

What are the 3 ways a DNA fragment can be produced?

Answer 14

• Reverse transcriptase
• Restriction endonucleases (enzymes)
• Gene machine

Question 15

How does reverse transcriptase produce a DNA fragment?

Answer 15

1) Extract and isolate mRNA from cell
2) T primers are added to the end of the mRNA
3) Reverse transcriptase is used to add free nucleotides to make a new strand of cDNA
4) New cDNA/mRNA strand is hydrolysed
5) DNA polymerase is added which synthesises double stranded DNA from the cDNA strand

Question 16

How do restriction endonuclease (enzymes) produce a DNA fragment?

Answer 16

1) Restriction enzymes cut at specific palindromic sequences (recognition sequences)
2) When recognition sequences are present restriction enzymes cut the DNA fragment out by hydrolysis
3) The fragment cut can leave either blunt or sticky ends
4) Sticky ends can bind to a complimentary sticky end to form new DNA fragment

Question 17

What are sticky ends?

Answer 17

Small tails of unpaired bases at each end of fragment after restriction enzymes have cut DNA

Question 18

What is a palindrome sequence?

Answer 18

Antiparallel base pairs that reads the same in opposite directions

Question 19

How does the gene machine produce a DNA fragment?

Answer 19

1) Sequence that is required is designed (if doesn’t already exist)
2) First nucleotide in sequence is fixed to some sort of support
3) Nucleotides are added step by step in correct order
4) Protecting groups make sure nucleotides join at right points to stop unwanted branching
5) Oligonucleotides (20 nucleotides long) are made and broken off from the support
6) Oligonucleotides are then joined together to make longer DNA fragments

Question 20

What are the positives of using a gene machine?

Answer 20

• Quick and accurate
• Easily transcribed in prokaryotic cells
• No introns or exons

Question 21

What are the 2 ways DNA can be amplified?

Answer 21

• In vivo amplification (vector)

- In vitro amplification (PCR)

Question 22

What are the 3 main stages of in vivo amplification?

Answer 22

1 - DNA fragment inserted into a vector
2 - Vector transfers DNA fragment into host cell
3 - Identify the transformed host cells

Question 23

How is the DNA fragment inserted into a vector (stage 1 of in vivo amplification)?

Answer 23

1) DNA fragment inserted into vector (plasmids or bacteriophages)
2) Vector cut by restriction enzymes so sticky ends are complementary to sticky ends on DNA fragment
3) Vector DNA and fragment DNA are mixed together with DNA ligases to form recombinant DNA (this is called ligation)

Question 24

How is the recombinant DNA inserted into the host cell using a plasmid (stage 2 of in vivo amplification)?

Answer 24

1) Host cells have to be persuaded to take in plasmid vector and its DNA
2) The host cells are placed in ice-cold calcium chloride which makes them more permeable
3) Plasmids are added and mixture is heat-shocked (42 degrees) which encourages cell to take up plasmids

Question 25

How is the recombinant DNA inserted into the host cell using a bacteriophage (stage 2 of in vivo amplification)?

Answer 25

1) The bacteriophage will infect the host bacterium by injecting its DNA into it
2) The phage DNA then integrates with the bacterial DNA

Question 26

What % of host cells with take up the vector and its DNA?

Answer 26

5%

Question 27

What are transformed cells?

Answer 27

Host cells that take up the vector

Question 28

How are transformed cells identified?

Answer 28

Marker genes

Question 29

When are marker genes inserted into the transformed cells?

Answer 29

Inserted into at the same time the gene is to be cloned so all transformed cells have the gene to be cloned and the marker gene

Question 30

What are the 2 ways marker genes can be detected?

Answer 30

• Using antibiotic resistance

- Fluorescence

Question 31

How can a marker gene be detected using antibiotic resistance?

Answer 31

Host cells are grown on agar plates containing specific antibody antibiotic - so only transformed cells with marker genes will grow and survive

Question 32

How can a marker gene be detected using fluorescence?

Answer 32

When agar plate is under UV light only transformer cells will turn fluorescent

Question 33

What do you need in order for transformer host cells to produce the protein coded for by DNA fragment?

Answer 33

Vector contains specific promoter and terminator regions

Question 34

What are promoter regions?

Answer 34

DNA sequences that tell RNA polymerase where to start producing mRNA

Question 35

What are terminator regions?

Answer 35

DNA sequences that tell RNA polymerase where to stop producing mRNA

Question 36

What is in vitro amplification?

Answer 36

Where copies of DNA fragments are made outside organsim using a polymerase chain reaction

Question 37

How does the polymerase chain reaction work (in vitro amplification)?

Answer 37

1) Reaction mixture is heated to 95 degrees to break hydrogen bonds between 2 strands
2) Mixture then cooled between 50-60 degrees so primers can bind to stands
3) Mixture then heated to 72 degrees so DNA polymerase can free nucleotides can join to form new strands
4) 2 new copies of fragment DNA are formed

Question 38

What is the reaction mixture in the PCR made of?

Answer 38

• DNA sample
• Primers
• Free nucleotides
• DNA polymerase

Question 39

How many strands would be made for each cycle of PCR?

Answer 39

1st cycle - 4
2nd cycle - 8
3rd cycle - 16
4th cycle - 32

Question 40

What are primers?

Answer 40

Short pieces of DNA that are complementary to start of fragment

Question 41

What is genetic engineering?

Answer 41

Where microorganisms, plants and animals are transformed using recombinant technology