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AS and A level > Biology A2 Chapter 21 - Recombinant DNA technology > Flashcards

Biology A2 Chapter 21 - Recombinant DNA technology Flashcards

1
Q

Define recombinant DNA

A

When the DNA of two different organisms has been combined

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2
Q

What is a transgenic/genetically modified organism?

A

One which has recombinant DNA

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3
Q

What are the stages of making a protein using the DNA technology of gene transfer and cloning? Give a brief explanation of each

A
  1. Isolation - isolate the DNA fragments that have the gene for the desired protein
  2. Insertion - insert the DNA fragment into a vector
  3. Transformation - transfer the DNA into suitable host cells
  4. Identification - identify the host cells that have successfully taken up the gene by the use of gene markers
  5. Growth/cloning - clone the population of host cells
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4
Q

What are the three methods of producing DNA fragments?

A
  • Conversion of mRNA to cDNA using reverse transcriptase
  • Using restriction endonucleases to cut fragments containing the desired gene from DNA
  • Create the gene in a gene machine
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5
Q

Describe the process of using reverse transcriptase to isolate a gene using insulin as an example

A
  1. B-cells from the islets of Langerhans in the human pancreas are required as they are specialised to produce insulin and so make a lot of mRNA which codes for insulin
  2. The mRNA acts as a template on which a single stranded complementary copy of DNA (cDNA) is formed using reverse transcriptase
  3. cDNA is isolated by the hydrolysis of the mRNA with an enzyme
  4. Double stranded DNA is formed on the cDNA template using DNA polymerase
  5. A copy of the human insulin gene is produced
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6
Q

Describe the process of using reverse transcriptase to isolate a gene

A
  1. A gene which readily produces the protein is selected
  2. These have large quantities of the relevant mRNA and so it is easily extracted
  3. Reverse transcriptase is used to make DNA from RNA. This is known as cDNA
  4. DNA polymerase forms the double strand of DNA required
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7
Q

What is a recognition sequence?

A

The particular sequence of bases where a restriction endonuclease cuts

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8
Q

What are the two ways in which restriction endonucleases cut DNA?

A
  • Blunt ends - cut to leave two straight edges, cut between two opposite base pairs
  • Sticky ends - an even cut where each strand of the DNA has exposed bases
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9
Q

Describe the process of using the gene machine to isolate a gene

A
  1. The desired sequence of bases is determined from the protein. The mRNA codons are worked out from the amino acid sequence and complementary DNA triplets are also worked out
  2. The desired base sequence is fed into a computer
  3. This is checked for biosafety and biosecurity reasons
  4. The computer designs a series of small, overlapping single strands of nucleotides, called oligonucleotides, which can be assembled into the desired gene
  5. This gene is then replicated using using PCR which also contracts the complementary stand of nucleotides to make the double stranded gene. This is then multiplies to give numerous copies
  6. Using sticky ends the gene can be inserted into a plasmid. This acts as a vector for the gene, allowing it to be stored, cloned, or transferred into other organisms in the future
  7. The genes are checked using standard sequencing techniques and those with errors are rejected
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10
Q

What is meant by in vivo?

A

Transferring the fragments into a host cell using a vector (in life)

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11
Q

What is meant by in vitro?

A

Using the polymerase chain reaction (PCR), so it is done ‘in glass’

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12
Q

What is the purpose of the enzyme DNA ligase?

A

Binds the phosphate sugar framework of the two sections of DNA to unite them as one

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13
Q

Why are sticky ends important?

A

Provided the same restriction endonuclease is used, we can combine the DNA of one organism with that of any other organism

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14
Q

Define the term promoter

A

The region of DNA which acts as a binding site for RNA polymerase and transcriptional factors, starting transcription

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15
Q

Define the term terminator

A

The region of DNA which releases RNA polymerase, ending transcription

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16
Q

Define the term vector

A

The carrying unit to which the DNA fragment is added

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17
Q

What is a vector used for?

A

To transport the DNA into a host cell

18
Q

Why will not all the bacterial cells possess the DNA fragment with the desired gene for the desired protein?

A
  • Only few bacterial cells (1%) take up the plasmids when the two are mixed together
  • Some plasmids will have closed up again without incorporating the DNA fragment
  • Sometimes the DNA fragment ends join together to form its own plasmid
19
Q

How do we find out which bacterial cells have taken up the plasmid containing the DNA fragment? What is the problem with this?

A
  1. All the bacterial cells are grown on a medium that contains the antibiotic ampicillin
  2. Bacterial cells that have taken up the plasmid will have acquired the gene for ampicillin resistance and so they can break down the ampicillin and survive
  3. The bacterial cells which have not taken up the plasmid will not be resistant and therefore will die
    The problem is that some cells which have taken up the plasmids which closed before incorporating the new gene, and these would also have survived
20
Q

How do we use marker genes to identify whether a gene has been taken up by bacterial cells?

A

We use a second, separate gene on the plasmid

21
Q

Why may certain genes on plasmids be easily identifiable using marker genes?

A
  • May be resistant to an antibiotic
  • May make a fluorescent protein which is easily seen
  • May produce an enzyme whose action can be identified
22
Q

How do we identify cells with plasmids that have taken up the desired gene?

A

Replica plating. The gene was cut to add the DNA fragment and in doing so it lost its resistance to tetracycline as it no longer produces the enzyme to break it down. This means they do not survive against tetracycline and so we can identify living colonies of bacteria containing the desired gene

23
Q

How do fluorescent markers work?

A

A gene from a jellyfish is transferred into a plasmid. This produces a green fluorescent protein (GFP). Any plasmid that has taken up the plasmid with the desired gene will not produce GFP and those will did not will produce GFP

24
Q

How do we use enzyme markers to identify which cell has taken up the desired gene?

A

If a plasmid with the required gene is present in a bacterial cell, the colonies grown will not produce lactase. Therefore, they will not turn from colourless to blue, and will remain colourless. This is because the required gene to produce lactase was removed from the plasmid when the DNA fragment was inserted

25
Q

What is the PCR useful for?

A

Making many copies of DNA fragments

26
Q

What does PCR require?

A
  • The DNA fragment
  • DNA polymerase
  • Primers
  • Nucleotides
  • Thermocycler
27
Q

What are primers?

A

Short sequences of nucleotides that have a set of bases complementary to those at one end of each of the DNA fragments

28
Q

What is are the three stages of the polymerase chain reaction?

A
  1. Separation of the DNA strand - the DNA fragments, primers, and the DNA polymerase are placed in a vessel in the thermocycler at 95 degrees which causes the two strands of DNA fragments to separate due to the breaking of hydrogen bonds
  2. Addition (annealing) of the primers - the mixture is cooled to 55 degrees so the primers can join (anneal) to their complementary bases at the end of the DNA fragment, providing starting sequences for DNA polymerase
  3. Synthesis of DNA - temperature raised to 72 degrees as this is the optimum temperature for DNA polymerase to add complementary nucleotides along each of the separated DNA strands
29
Q

What are the advantages of in vitro gene cloning?

A
  • Extremely rapid
  • Does not require living cells
30
Q

What are the advantages of in vivo cloning?

A
  • Particularly useful where we wish to introduce a gene into another organism
  • Involves almost no risk of contamination
  • Very accurate
  • Cuts out specific genes
  • Produces transformed bacteria that can be used to produce large quantities of gene products
31
Q

What is the process for locating specific alleles for genes?

A
  1. We must first determine the sequence of nucleotide bases of the mutant allele we are trying to locate. This can be done via the use of genetic libraries
  2. A fragment of DNA is produced that has a sequence of bases which are complementary to the mutant allele we are trying to locate
  3. Multiple copies are formed using PCR
  4. A DNA probe is made by attaching a marker, like a fluorescent dye, to the DNA fragment
  5. DNA from the person with the suspected mutant allele is heated to separate strands
  6. The separated strands are cooled in a mixture containing many DNA probes
  7. If the mutant allele is present, the probe binds
  8. The DNA is washed clean of nay unattached probes
  9. The remaining hybridised DNA will now be fluorescently labelled which is detected by shining a light onto the fragment, causing the dye to fluoresce which can be seen using a special microscope
32
Q

What are variable number tandem repeats (VNTRs)?

A

DNA bases which are non-coding

33
Q

What is gel electrophoresis used for?

A

The separation of DNA fragments according to their size

34
Q

What are the 5 steps (basic) of genetic fingerprinting?

A
  1. Extraction
  2. Digestion
  3. Seperation
  4. Hybridisation
  5. Development
35
Q

How does genetic fingerprinting work? Give the process

A
  1. DNA is extracted from the sample
  2. Restriction endonucleases cut the DNA into fragments
  3. Fragments are separated using gel electrophoresis
  4. DNA fragments are transferred from the gel to a nylon membrane
  5. DNA probes are added to label the fragments. These radioactive probes attach to specific fragments
  6. The nylon membrane with radioactively labelled DNA fragments is placed onto an X-ray film
  7. Development of the X-ray film reveals dark bands where the radioactive DNA probes have attached
36
Q

How does gel electrophoresis work?

A

The DNA fragments are placed on an agar gel and a voltage is applied across it. The resistance of the gel means that the larger the fragments, the more slowly they move. Therefore, over a fixed period, the smaller fragments move further than the larger ones

37
Q

What can genetic fingerprinting be used for?

A
  • Forensic science
  • Paternity testing
  • Medical diagnosis
  • Plant and animal breeding
38
Q

What evidence can be used for genetic fingerprinting at a crime scene?

A

Blood, semen, hair follicles

39
Q

Even if there is a close match between the DNA of the suspect and the DNA at a crime scene, this does not prove they carried out the crime. Why?

A
  • The DNA may have been left on some other, innocent occasion
  • The DNA may belong to a very close relative
  • The DNA sample may have been contaminated after the crime, either by the suspects DNA or chemicals which affected the action of restriction endonucleases used in preparing the fingerprint
40
Q

What diseases can genetic fingerprinting help to diagnose and how?

A

Huntington’s disease. It results from the sequence AGC on the end of a gene on chromosome 4 being repeated over and over again. A sample of DNA from a person with the allele for the disease can be cut with restriction endonucleases and a DNA fingerprint prepared. This can be compared with people with various forms of he disease and those without the disease so the probability of developing symptoms, and when, can be determined

41
Q

How is genetic fingerprinting used in plant and animal breeding?

A

It can be used to prevent undesirable inbreeding due to seeing if the genetic fingerprints are close matches, and can also be used to identify plants or animals which possess a particular allele for a desired gene

AS and A level (30 decks)

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