Gene Tech (All cards)

Question 1

What is in vivo cloning?

Answer 1

(In the body)

Transferring the fragments of DNA to a host cell using a vector

Question 2

What does DNA ligase do?

Answer 2

Bind two bases that have lined up with sticky ends together

Question 3

Why are restriction enzymes important?

Answer 3

Can cut DNA so that all fragments will have the same complementary ends.

Question 4

What do promoter regions do?

Answer 4

Tell RNA polymerase where to start.

Question 5

What do terminator genes do?

Answer 5

Tell RNA polymerase where to start

Question 6

What happens in the Isolation stage of in vivo cloning?

Answer 6

• DNA fragments are produced using restriction endonuclease.
• Promoter regions are added so that RNA polymerase and transcription factors can attach and begin transcription.
• Terminator regions are added to detach RNA polymerase.

Question 7

What during the Insertion stage of in vivo cloning?

Answer 7

• DNA fragment is inserted into a vector
• This is used to transport the DNA into the host cell.
• Plasmids are commonly used - these are circular lengths of DNA, found in bacteria, a d contain genes for antibiotic resistance.
• The same restriction endonucleases are used at one of these ABR genes to break the plasmid loop.
• DNA fragments mix with the opened up plasmid and become incorporated and the join is made permanent using DNA ligase.

Question 8

Why is the same restriction endonuclease enzyme used to break the plasmid loop?

Answer 8

Ensures that the sticky ends of the opened-up plasmid are complementary to the sticky ends of the DNA fragment.

Question 9

What happens during the Transformation stage of in vivo cloning?

Answer 9

• DNA fragment is mixed in a medium containing calcium ions and a change in temperature is brought which make the bacterial membrane permeable.
• This allows the plasmids to pass through the cell-surface membrane into the cytoplasm.

Question 10

What happens during the identification stage of in vivo cloning?

Answer 10

• Marker genes are used are used to identify whether a gene has been taken up by bacterial cells.
  There a three types:
• Antibiotic resistant genes
• May make an easy to see fluorescent protein
• May produce an enzyme

Question 11

How are antibiotic resistant marker genes used?

Answer 11

• These bacteria can be grown on a culture containing an antibiotic.

Question 12

How are fluorescent marker genes used in in vivo cloning?

Answer 12

Bacterial cells that have not taken up the gene will not fluoresce.

Question 13

How do enzyme markers in in vivo cloning work?

Answer 13

• This gene produces lactase which turns particular substrates blue.
• So when these bacterial cells are grown on a medium with these particular substrates, they will turn blue.

Question 14

what is in vitro cloning?

Answer 14

(Outside the body)

Using PCR

Question 15

What is the purpose of PCR?

Answer 15

To copy DNA fragments

Question 16

What does PCR require?

Answer 16

• DNA fragment to be copied
• TAQ polymerase: Resistant to high temperatures, used to join together nucleotides
• Primers
• Nucleotides

Question 17

What are the stages of PCR?

Answer 17

• Separation of the DNA strand
• Addition of primers
• Synthesis of DNA

Question 18

What happens during the separation stage of PCR?

Answer 18

• Temperature increases to 95°C causing the double helix DNA strand to separate due to the breaking of hydrogen bonds,

Question 19

What happens in the addition stage of PCR?

Answer 19

• Mixture is cooled to 55°C, causing the primers to anneal (join) to their complementary bases.
• Primers provide the starting sequence for DNA polymerase.
• Also prevent the two separate strands from re-joining.

Question 20

What happens during the synthesis stage of PCR?

Answer 20

• Temp increases to 72°C.
• Optimum temp for DNA polymerase to add complementary nucleotides along each separated DNA strand until it reaches the end strand.

Question 21

What happens after PCR?

Answer 21

Process repeats

Question 22

Advantages of in vitro gene cloning

Answer 22

• Many copies of DNA can be made in a short amount of time but also contaminate DNA
• Only samples are required, no living organism

Question 23

Advantages of in vivo cloning

Answer 23

• Gene therapy, useful when we want to introduce a gene into a living organism
• Almost no risk of contamination
• DNA copied is very accurate
• Precisely cuts out specific genes
• Can be used to produce large quantities of gene products, commercial and medical use

Question 24

What are the ways that DNA fragments can be produced?

Answer 24

• Reverse transcriptase
• Restriction endonuclease
• Gene Machine

Question 25

How is reverse transcriptase used to form DNA fragments?

Answer 25

- mRNA coding for the desired gene is isolated. - Reverse transcriptase is used to form cDNA by using mRNA as a template. - mRNA detaches from cDNA through hydrolysis using an enzyme. - DNA polymerase is used on the template cDNA to form double stranded DNA. - This has a copy of the desired gene.

Question 26

What is cDNA?

Answer 26

Single stranded complementary copy of DNA

Question 27

How are restriction enzymes used to produce DNA fragments?

Answer 27

- Cut a DNA double strand at recognition sites.

Question 28

Where does reverse transcriptase come from?

Answer 28

Retroviruses

Question 29

How does the gene machine produce DNA fragments?

Answer 29

- The desired sequence of DNA is worked out from a database. | - The mRNA codons are found and the complementary DNA is worked out.

Question 30

What are DNA probes?

Answer 30

Short, single stranded lengths of DNA that can be easily identified

Question 31

What are the types of DNA probes?

Answer 31

- Radioactive probes: identified using X-Ray | - Fluorescent probes: fluoresce under certain conditions

Question 32

How do DNA probes work?

Answer 32

- The double stranded DNA is separated. - Separated strands are mixed with the probe - The probe has base sequences that are complementary to part of the base sequence of the DNA (coding for the allele) that we want to find so DNA hybridisation occurs. - This site is then identified (using radioactivity or fluorescence)

Question 33

What is DNA hybridisation?

Answer 33

When a section of DNA/RNA binds with a single section of

Question 34

What is recombinant DNA?

Answer 34

DNA from two different organisms that have combined.

Question 35

What is the purpose of gene screening?

Answer 35

- This allows for multiple genetic disorders to be screened for at the same time

Question 36

What happens in the screening of genes?

Answer 36

1) DNA sample is washed over the array. 2) If the sample contains the target gene, it will attach to the probe (on the array). 3) Array is washed to remove any unattached DNA. 4) Array is placed under UV light. 5) Spots that fluoresce indicate that the mutated allele is present.

Question 37

What are the types of Gene Therapy?

Answer 37

Somatic Therapy | Germ line Therapy

Question 38

What is Somatic Gene Therapy?

Answer 38

- Altering the alleles in the body cells. | - Does not affect the individual sex cells.

Question 39

What is Germ Line Therapy?

Answer 39

- Involves altering the alleles in sex cells.

Question 40

What are VTNRs?

Answer 40

Variable Number Tandom Repeats | - Sequences that don't code for proteins and are repeated.

Question 41

How is a genetic fingerprint made?

Answer 41

1) Obtain a DNA sample 2) Use PCR to make copies 3) Cut into fragments using restriction endonuclease 4) Fragments are separated according to size using gel electrophoresis 5) Then transferred to nylon membrane 6) DNA probes are added to label specific fragments to be identified

Question 42

What is gel electrophoresis?

Answer 42

Separates DNA fragments by size: - Fragments are placed onto an agar gel and an electrical current is passed through. - Larger the fragments, the slower they move. Therefore smaller fragments move further in a fixed time. - They can be tracked using probes