Gene Technology

Question 1

What are the 3 ways we can obtain gene fragments?

Answer 1

Restriction endonuclease
Reverse transcriptase
Gene Machine

Question 2

What are the 2 ways we can use restriction endonuclease to obtain gene fragments?

Answer 2

1. Cuts DNA on a palindromic sequence ( sequence with antiparallel base pairs) creating sticky ends
2. Cuts DNA on variable nucleotide tandem repeats

Question 3

How do we use reverse transcriptase to obtain gene fragments?

Answer 3

Converts mRNA to double stranded cDNA - this does not have introns or promoter regions

Question 4

How do we use a gene machine to obtain gene fragments?

Answer 4

DNA synthesiser - can make short sequences of DNA

Question 5

How can we amplify genes?

Answer 5

Via in vivo and in vitro techniques

Question 6

What is recombinant DNA technology?

Answer 6

Transferring DNA fragments from one organism to another, creating transgenic organisms with combined genetic material

Question 7

How do we obtain the desired gene in in vivo techniques?

Answer 7

Cut the gene with the restriction endonuclease, creating sticky ends

Question 8

How do we prepare the vector to be combined with the DNA in in vivo techniques?

Answer 8

Cut the plasmid/vector with the same enzyme (as was used for the gene), creating complementary sticky ends

Question 9

How are the desired gene and the vector combined in in vivo techniques?

Answer 9

DNA ligase is used to create phosphodiester bonds, binding the gene into the plasmid/vector
(they have complementary sticky ends)

Question 10

How do we identify the bacteria has taken up the plasmid with the recombinant DNA in in vivo techniques?

Answer 10

Add a marker gene and incubate the plasmid with the bacteria
(for the marker gene, either use antibiotic resistance or GFP - green fluorescence protein- or radioactivity)

Question 11

How are the bacteria with the plasmids identified in in vivo techniques after the marker gene has been added?

Answer 11

Incubate bacteria - those with the marker genes will either survive antibiotic/glow under UV/produce radiation - autoradiography can see which have it

Question 12

What do we use to genetically modify an animal using in vivo techniques?

Answer 12

We use a virus (lentivirus) to insert DNA into the host cell genome - of the embryo

Question 13

What do we use to genetically modify a plant using in vivo techniques?

Answer 13

We can use agrobacterium and put the gene into the seed

Question 14

Model 6 marker for PCR question?

Answer 14

1. DNA heated to 90-95
2. Strands separate as hydrogen bonds break
3. Cooled to 55
4. Add Primers and they bind
5. Nucleotides bind by complementary base pairing
6. heat back to 72
7. DNA polymerase joins nucleotides together via phosphodiester bonds
8. cycle repeated

Question 15

What are the 2 types of gene therapies?

Answer 15

Somatic therapy
Germ line therapy

Question 16

What is somatic therapy?

Answer 16

This involves altering the alleles in body cells - particularly the cells that are most affected by the disorder

Question 17

What does somatic therapy not affect?

Answer 17

The individual’s sex cells - so any offspring could still inherit disease

Question 18

What is germ line therapy?

Answer 18

Involves altering the alleles in sex cells - every cell of any offspring will be affected by the gene therapy and they won’t suffer from the disease
(this therapy in humans is currently illegal)

Question 19

What does PCR stand for?

Answer 19

Polymerase chain reaction

Question 20

What do we add to the test tube for PCR?

Answer 20

Original DNA sample, primers, heat resistant DNA polymerase, nucleotides

Question 21

What is the first step of a PCR test?

Answer 21

Heat DNA sample to 95 degrees to separate DNA strands - breaking hydrogen bonds between strands

Question 22

What do we do in the PCR test after separating the DNA sample into separate strands?

Answer 22

Cool to 55 degrees to allow primers to anneal

Question 23

What are primers?

Answer 23

21 nucleotide long sequences of complementary single stranded DNA

Question 24

What is the purpose of primers?

Answer 24

• Bind to DNA to allow DNA polymerase place to start replicating
• Marks start and end of DNA sequence to be copied i.e. to only amplify DNA you are researching

Question 25

What is annealing in PCR?

Answer 25

This is allowing the primers to complementary base pair to DNA strands

Question 26

What is the next step in PCR testing after the primers have annealed to the sample?

Answer 26

Heat to 72 degrees for DNA polymerase to create phosphodiester bonds

Question 27

How do you create multiple DNA samples in PCR?

Answer 27

Repeat the process many times

Question 28

What is genetic sequencing?

Answer 28

Working out the exact base sequence for a gene (the sequence of nucleotides) Much faster nowadays

Question 29

What kind of gel do we use for electrophoresis?

Answer 29

A polyacrylamide gel

Question 30

How is DNA added to the gel in electrophoresis?

Answer 30

Using a polyacrylamide gel, DNA is added to a buffer that makes it negatively charged

Question 31

What causes DNA to move in electrophoresis?

Answer 31

Putting a current across the gel causes the DNA to move towards the positive electrode/anode

Question 32

What length DNA will take longer to move through the gel?

Answer 32

DNA that is longer - so the longer stands won't have moved as far as shorter strands

Question 33

What is DNA separated based on in electrophoresis?

Answer 33

Based on length/charge

Question 34

For genetic fingerprinting, how do we obtain the DNA we want to look for?

Answer 34

DNA is cut using restriction endonuclease at VNTRs (PCR is used to make a lot of the DNA fragments)

Question 35

What do we use in genetic fingerprinting to separate the DNA fragments?

Answer 35

Use electrophoresis - separates according to length/mass

Question 36

After electrophoresis in genetic fingerprinting, where do we transfer the DNA fragments to?

Answer 36

To a nylon membrane

Question 37

After electrophoresis, how do we separate the DNA in genetic fingerprinting?

Answer 37

Make it single stranded by immersing gel in an alkaline solution

Question 38

How do we identify the DNA in genetic fingerprinting after transferring it to a nylon membrane?

Answer 38

Apply probe that's either radioactive of fluorescent: -Radioactive - use autoradiography - Fluorescent - use UV light

Question 39

How do we see DNA bands on an electrophoresis gel?

Answer 39

We need to add a DNA probe

Question 40

What is a DNA probe?

Answer 40

A single stranded complementary DNA with either UV sensitive marker of a radioactive isotope attached

Question 41

What do we have to do to the DNA bands before adding a DNA probe?

Answer 41

Make it single stranded

Question 42

After adding the DNA probe, how is the DNA identified?

Answer 42

If UV sensitive marker - use UV light If radioactive isotope used - use autoradiography

Question 43

If you are looking for a specific mutation in DNA, what are the steps to identify it?

Answer 43

- Create DNA probe complementary to mutation - Add to the nylon membrane after electrophoresis - Will bind to the DNA if mutation is present - So will glow/produce radioactivity at the distance we expect for the length of DNA we suspect the mutation will be