19.1. Principles of Genetic Technology

Question 1

Aim of Genetic Technology / Engineering

Answer 1

• to remove a gene (or genes) from one organism and transfer it into another so that the gene is expressed in its new host.
• The DNA that has been altered by this process and which now contains lengths of nucleotides from two different organisms is called recombinant DNA (rDNA).
• The organism which now expresses the new gene or genes is known as a transgenic organism or a genetically modified organism (GMO).

Question 2

Recombinant DNA

Answer 2

DNA made by joining pieces from two or more different sources.
- result of introduction of DNA from a different species

Question 3

Polymerase Chain Reaction (PCR)

Answer 3

• a technique that is used to amplify one sample of DNA thousands of times over to create a large enough DNA sample for extensive analysis
• I.e. each time a PCR cycle is performed the total amount of DNA is doubled
• It is In Vitro (in glass) Amplification of DNA

Question 4

Uses of PCR

Answer 4

1) Paternity tests - The child’s tandem repeats are compared to the mother’s and the father’s
2) Detecting mutations - comparing one person’s DNA to the DNA of the person with the mutation
3) Increasing small quantities of DNA from crime scenes so that it may be analysed

Question 5

Steps of PCR

Answer 5

1) Denature the DNA
2) Anneal the DNA
3) Extension of DNA

Question 6

Denaturing the DNA in the process of PCR

Answer 6

• “Denature” means to separate the DNA strands into 2 separate strands (breaking H-H bonds)
• This involves heating the DNA sample up to 95 degrees!

Question 7

Annealing the DNA in the process of PCR

Answer 7

• “Annealing” means to add
• Primer binding (connecting)
• In this step 2 primers are added to the 2 separated DNA strands (1 on each strand)
• The temperature is “cooled” to 65 degrees, this helps the primers bind to the DNA

Question 8

Extension of DNA in the process of PCR

Answer 8

• New DNA created
• 2 polymerase molecules attach to the 2 primers on the 2 DNA strands and move along the strand
• The polymerase molecules are called Taq Polymerase which were sourced from a bacterium living in hot springs which are stable even at high temperatures
• As they move along they create new “complementary” DNA
• Temperature goes up to 72 degrees

Question 9

Advantages of using PCR

Answer 9

• Only need a small sample of DNA
• Quickly amplify the DNA sample
• Very efficient process
• Taq Polymerase does not need replacing

Question 10

Uses of Gel Electrophoresis

Answer 10

A way of separating:

• strands of DNA of different lengths
• polypeptides with different base sequences

Question 11

Gel Electrophoresis Process

Answer 11

• Before electrophoresis is carried out, the sample of DNA is exposed to a set of restriction enzymes (restriction endonucleases) which cut DNA molecules where particular base sequences are present
• This cuts the DNA into fragments of different lengths
• To carry out gel electrophoresis, a small shallow tank is filled with a layer of agarose gel. An electrical potential difference is applied across the gel, so that a direct current flows through it
• DNA fragments are placed in wells of the gel
• DNA fragments have a negative charge due to its negatively charged phosphate group, so these fragments move from the cathode (negative) towards the anode (positive)
• The smaller the fragments, the faster they move.
• To make DNA visible, fluorescent markers are added to the fragments
• Alternatively, single strands of DNA can be made using radioactive isotopes, and with base sequences thought to be similar to those in DNA, can be added to gel which are called probes
• They will pair up with fragments that have complementary base sequences so their positions are now emitting radiation
• This can be detected by its effects on a photographic plate

Question 12

Application of Gel Electrophoresis

Answer 12

1) Distinguishing between polypeptides or proteins
2) Distinguishing between different alleles of a gene
3) Genetic Fingerprinting

Question 13

Distinguishing between polypeptides or proteins using Gel Electrophoresis

Answer 13

• polypeptides are made up of long chains of amino acids which differ in the charge they carry, because different R groups have different charges
• the differences mean that the polypeptides would move at different speeds on the electrophoresis gel
• etc. normal B polypeptide has a charged R group whereas sickle cell B polypeptide has an uncharged R group

Question 14

Distinguishing between different alleles of a gene using Gel Electrophoresis

Answer 14

• DNA strands containing different alleles of a gene may end up at different places on the gel after electrophoresis
• For example, one allele may have more bases than another, and so be more massive and move more slowly

Question 15

Genetic Fingerprinting using Gel Electrophoresis

Answer 15

• some regions of DNA are very variable, containing different numbers of repeated DNA sequences
• these are known as variable number tandem repeats (VNTRs)
• each person’s set of VNTR sequences is unique, but identical twins share identical VNTR sequences
• restriction enzymes are used to cut a DNA sample near VNTR regions and the chopped pieces of DNA are separated using gel electrophoresis
• long VNTR sequences don’t travel as far as short ones
• the pattern of stripes produced is therefore determined by the particular combinations that a person has
• genetic fingerprinting can determine whether a sample of semen, blood or other tissue found at a crime scene could have come from a victim or suspect, or whether a particular person could be the child, mother or father of another

Question 16

Genetic Engineering Steps

Answer 16

1) Isolation - of the DNA containing the required gene
2) Insertion - of the DNA into a vector
3) Transformation - Transfer of DNA into a suitable host
4) Identification - finding those host organisms containing the vector and DNA (by use of gene markers)
5) Growth / cloning - of the successful host cells

Question 17

Isolation in Genetic Engineering

Answer 17

For example: Insulin

• B-cells from Islets of Langerhans in the Human pancreas.
• Extract mature mRNA coding for Insulin.
• A single stranded complementary copy of DNA (cDNA) is formed using reverse transcriptase on the mRNA template.
• Single stranded cDNA is used to form double stranded DNA using DNA polymerase
• This forms a double stranded copy of the Human Insulin gene.

Question 18

Reverse Transcriptase Function

Answer 18

• A group of viruses called retroviruses (e.g. HIV) contain an enzyme called reverse transcriptase
• It is used to turn viral RNA into DNA so that it can be transcribed by the host cell into proteins
• Reverse Transcription makes DNA from an RNA template - it does the opposite of transcription

Question 19

Insertion in Genetic Engineering

Answer 19

• DNA from different sources can be joined together IF they have the same sticky ends - same restriction sites
• In order to have the same restriction sites, they must have been cut using the same restriction endonuclease
• DNA is inserted into a vectors like a plasmid
• Sticky ends are joined together using DNA ligase to join the sugar phosphate backbone together
• The new DNA molecule is called recombinant DNA

Question 20

Sticky ends

Answer 20

• Bacteria contain restriction enzymes (restriction endonuclease) in order to protect themselves from invading viruses.
• Restriction enzymes are used by bacteria to cut up the viral DNA.
• These enzymes cut DNA at specific sites – this property can be useful in gene technology.
• DNA can be cut to form Blunt Ends or Sticky Ends
• Blunt ends are cut straight through DNA
• Most enzymes make a staggered cut in the two chains, forming sticky ends with a single strand of DNA which are complementary to one another

Question 21

Transformation in Genetic Engineering

Answer 21

• The plasmids must be reintroduced into the host cell e.g. bacteria
• This process is called transformation.
• The bacteria, plasmids and calcium are mixed together.
• By altering the temperature the bacteria become permeable and the plasmid can pass through the cell membrane.
• This can also be done by electroporation where a small electric current is used to make the membrane more porous

Question 22

Identification in Genetic Engineering

Answer 22

• Gene markers are used to identify which plasmids have taken up the DNA fragment.
• Usually the gene marker is disrupted if the DNA fragment is present.

Question 23

Types of Gene Markers

Answer 23

1) Fluorescent markers
2) Enzyme markers
3) Antibiotic-resistance markers

Question 24

Fluorescent Markers: a type of Gene Marker

Answer 24

• the gene from jellyfish which produces Green Fluorescent Protein (GFP) has been incorporated into a plasmid
• if the host cell has been successfully transformed it will glow under green light

Question 25

Enzyme markers: a type of Gene Markers

Answer 25

• The enzyme B-glucoronidase turns a colourless substance a blue colour
• If the gene has been disrupted by the incorporation of the gene fragment the substrate will remain colourless

Question 26

Antibiotic-resistance markers

Answer 26

• the antibiotic resistance gene is disrupted when the restriction enzyme cuts open the plasmid
• the second antibiotic-resistance gene (e.g. resistance
  to tetracycline) is used to identify those plasmids with
  a DNA fragment in them.
• If the DNA fragment has been inserted into the
  tetracycline resistance gene it will no longer grow on
  medium containing tetracycline.
• In order to identify these bacteria we use a process
  called replica plating.
• add antibiotics to replicas of the masterplate and try to identify the bacteria that survive

Question 27

Cloning in Genetic Engineering

Answer 27

• Following successful identification of the bacteria containing the plasmid AND the DNA fragment, the bacteria are cloned.
• As the bacteria are cloned, so is the plasmid containing the DNA fragment.
• This type of gene cloning is in vivo (cloned within a living organism).
• bacteria are grown in fermenters, where they are provided with nutrients and oxygen to allow them to reproduce to form large populations
• these GM bacteria are now being cultured on a large scale which synthesise and secret insulin, which is harvested by fermenters and purified before sale

Question 28

Vectors

Answer 28

a structure that transfers DNA into the organism that is to be modified.

vectors can be:

• plasmids - most suitable as vectors
• viruses - they naturally enter cells
• liposomes

Question 29

Why are Plasmids used?

Answer 29

• Found in bacteria = easily taken up
• Replicate independently in bacteria (origin of replication) = cloning
• Can be transferred between species of bacteria = flexibility
• Can be cut at specific locations = good for gene insertion
• Easy to extract from bacteria
• Genes can be inserted
• May contain antibiotic resistance genes

Question 30

Importance of Promoters in Genetic Engineering

Answer 30

• They are the ‘switch’ for the gene
• In bacteria, each gene is associated with a region of DNA called a promoter
• The enzyme RNA polymerase must bind to the promoter before it can begin transcribing the DNA to produce mRNA
• If a promoter is not included, the gene will need to be placed next to an existing one, which is very difficult!
• therefore it is important to ensure there was a promoter associated with the human insulin gene before inserting it into the bacteria E.Coli

Question 31

What is a Microarray

Answer 31

“gene chip”
- a tool used to identify partciular DNA sequences in a sample
- based on a small piece of glass usually 2cm^2 to which DNA probes have been attached to a regular pattern
- around 10 000 or more probes can be attached to one microarray
- the probes are from known locations across the
chromosomes of the organism involved

Question 32

Microarray Uses

Answer 32

1) Detect particular alleles

2) Analyse gene expression

Question 33

Microarrays in detecting particular alleles

Answer 33

• DNA is extracted and cut up into fragments by restriction enzymes to give ssDNA and denatured to give lengths of single-stranded DNA.
• DNA has a fluorescent label attached to it (usually red or green)
• The labelled DNA samples are mixed together and allowed to hybridise with the probes on the microarray.
• Any DNA that does not bind to probes on the microarray is washed of.
• microarray is then inspected using ultraviolet light, which causes the tags to fluoresce.
• Any positions that fluorescence contain can be used to identify the alleles present - DNA fragments are complementary to the probes.
• Microarray is then scanned so that the data can be read by a computer.
• Green and red fuorescent spots indicate where DNA from one species only has hybridised with the probes.
• Where DNA from both species hybridise with a probe, a yellow colour is seen.
• Yellow spots indicate that the two species have DNA with exactly the same base sequence, which suggests they have the same genes
• If there is no colour it means that no DNA has hybridised with the probe and that a particular gene is not present in either species.

Question 34

Microarrays in analysing gene expression

Answer 34

• used to compare which genes are active by identifying the genes that are being transcribed into mRNA
• mRNA from the two types of cell is collected and reverse transcriptase is used to convert mRNA to cDNA
• quantity of mRNA is small, so quantity of cDNA may
  need to be increased by PCR
• cDNA is labelled with fluorescent tags, denatured to give single-stranded DNA and allowed to hybridise with probes on the microarray.
• Spots on the microarray that fluoresce indicate the genes that were being transcribed in the cell.
• The intensity of light emitted by each spot indicates the level of activity of each gene.
• A high intensity indicates that many mRNA molecules were present in the sample, while a low intensity indicates that there were very few.
• The results therefore not only show which genes are active, but also their level of activity.

Question 35

Probe

Answer 35

Defined nucleic acid (DNA or RNA) that can be used to identify, usually through autoradiography, specific DNA or RNA molecules bearing the complementary sequence