Chapter 19- Genetic Technology

Question 1

Define genetic engineering

Answer 1

• Any procedure in which the genetic information in an organism is changed by:
  -altering the base sequence of a gene
  -introducing a gene from another organism
• The organism is said to be a generically modified organism

Question 2

Define recombinant DNA

Answer 2

This is DNA made by artificially joining together pieces of DNA from 2 or more different species or the same.

Question 3

Define transgenic organism

Answer 3

An organism that contains DNA from another source, such as from another individual of the same species or from a different species
- This is specific to interspecies gene transfer

Question 4

Define GMO

Answer 4

Any organism that has had its DNA changed in a way that does not occur naturally or by selective breeding
- This is open to all genetic modifications

Question 5

State the essential steps in which a GMO may be produced:

Answer 5

1- The gene that is required is identified and may be cut from a chromosome
2- Multiple copies of the gene are made using a technique called the polymerase chain reaction
3- The gene is inserted into a vector which delivers the gene to the cells of the organism.
4- The vector takes the gene into the cells
5- The cells that have the new gene are identified and cloned

Question 6

Define restriction enzymes

Answer 6

This is an enzyme, originally derived from bacteria, that cuts DNA molecules; each type of restriction enzyme cuts only at a particular sequence of bases.

Question 7

What are the restriction sites

Answer 7

• these are target sites that have specific sequences of 4 to 6 bases.
• Restriction enzymes either cut straight across the sugar-phosphate backbone to give blunt ends or they cut in a staggered fashion to give stick ends
  Sticky ends: Short lengths of unpaired bases

Question 8

How do you find the specific piece of DNA

Answer 8

• This involves separating the lengths of DNA using gel electrophoresis and gene probes.
• Gene probes is a single-stranded DNA with a known base sequence.
• It binds with lengths of DNA which have a complementary base sequence

Question 9

Describe reverse transcriptase

Answer 9

• This is an enzyme found in viruses that have RNA as their genetic material
• This enzyme uses single-stranded mRNA as a template and free DNA nucleotides to form a stranded DNA. This Is with the help of a DNA primer
• The process is complete when DNA polymerase synthesises another polynucleotide to form double-stranded complementary DNA (cDNA)
• The mRNA template is digested by RNA-ase enzyme.
• Sticky ends will then be added for insertion of cDNA into the plasmid

Question 10

Describe how synthesis of DNA can take place

Answer 10

• First, choose the codons for the amino acid sequence they need.
• The sequence of nucleotides is held in a computer that directs the synthesis of short fragments of DNA in DNA synthesiser machines
• These fragments are then joined together to make a longer sequence of nucleotides that can be inserted into plasmids for genetic engineering.

Question 11

Define vectors

Answer 11

These transport something from one place to another

Question 12

Describe how plasmids are obtained

Answer 12

• They treat bacteria with enzymes that break down their cell walls
• They then spin the bacteria at high speed in a centrifuge so that the larger circular DNA is separated from the much smaller plasmids
• The circular DNA of the plasmid is cut open using a restriction enzyme. The same enzyme is used to cut the gene that will be used

Question 13

State the advantages of using plasmids as vectors

Answer 13

• Their small size makes it easy to isolate and modify
• They can replicate independently and rapidly
• They can be used in many types of host cells
• Plasmids are stable in host cells

Question 14

Which enzyme links together the sugar-phosphate backbones of the DNA and the plasmids and how do they do it

Answer 14

DNA ligase!!!
It does this by catalysing the formation of phosphodiester bonds, forming a closed circle of double-stranded DNA containing the new gene.

Question 15

Other than plasmids, name 2 more vectors

Answer 15

• Viruses
• Liposomes

Question 16

Describe the steps to getting the plasmids into the bacteria (Transformation)

Answer 16

• First, the bacteria and plasmids are put into a solution with a high concentration of calcium ions
• The mixture is cooled and given a heat shock
• This will increase the chances of plasmids passing through the cell surface membrane of the bacteria. A small proportion of the bacteria (1%) take up plasmids with the gene. They are said to be transformed.

Question 17

Describe how to identify the bacteria with the recombinant DNA

Answer 17

• DNA polymerase in bacteria will copy the plasmids
• Bacteria will divide by binary fission so that each daughter cell has several copies of the plasmid (Gene cloning)
• The bacteria may also transcribe the new gene and use the mRNA in translation to form a protein. This is called a recombinant protein

Question 18

Describe how Recombinant human insulin is made step by step

Answer 18

• First the gene is cut out from mRNA for insulin from pancreatic B-cells.
• The mRNA is used as a template for reverse transcriptase to make single-stranded DNA
• The single stranded DNA molecules were then used as the template for DNA polymerase to make double-stranded DNA
• It will carry the insulin genes and will be inserted into the plasmids. The plasmids will then transform the bacterium E.coli

Question 19

Advantages of Genetic engineering insulin

Answer 19

+There is now a reliable supply available to meet the increasing demand.
+The insulin is human insulin not animal insulin
+Can modify the insuline gene for example making them act faster and

Question 20

Describe an example of genetic markers with fluorescent substances

Answer 20

• Enzymes obtained from jellyfish make a protein called GFP which fluoresces bright green ultraviolet light.
• The gene for the enzyme is inserted into the plasmids
• Now all that needs to be done is to identify the bacteria that have taken the plasmid by observing whether they shine ultraviolet light onto them.
• The ones that glow green are genetically modified.

Question 21

Describe the promoter

Answer 21

This is the region of DNA to which RNA polymerase binds as it starts transcription.-
- The promoter can be said to control the expression of a gene and ensure high level of gene expression
- The promoter allows RNA polymerase to bind to DNA and ensures that it recognises which of the 2 DNA strands is the template strand

Question 22

Define Gene editing

Answer 22

A form of genetic engineering in which the genome of an organism can be changed by deleting, inserting or replacing a length of DNA

Question 23

What is ‘Crispr’

Answer 23

This is a group of base sequences that code for short lengths of RNA that direct a nuclease enzyme known as Cas9 towards specific sequences.

Question 24

What is the role of Crispr-associated (Cas) enzyme

Answer 24

This is an endonuclease that cuts DNA at a point that is determined by these RNA molecules, known as guide RNA(gRNA)

• Part of gRNA has a sequence of 20 bases that can locate and bind to a strand of DNA with the complementary base sequence. The enzyme Cas9 has 2 active sites to cut DNA across both sides.
• This gives engineers a way to edit DNA at specific places within an organisms genome.

Question 25

What is the everyday use of Crispr/cas9

Answer 25

• Can be used to prepare customised drugs to treat cancers
• Can be used to edit the genomes of insect vectors to prevent the transmission of diseases such as malaria and viruses
• Can be used to target multiple genes at the same time

Question 26

Define the polymerase chain reaction

Answer 26

An automated process that amplifies selected regions of DNA using alternate stages of polynucleotide sepration (denaturation of DNA) and DNA synthesis catalysed by DNA polymerase

Question 27

List the following substances that are added to each tube in the PCR machine

Answer 27

• A sample of DNA
• 2 different short lengths of single-stranded DNA to act as primers for the DNA polymerase
• free dNTPs
• A buffer solution
• A solution of heat-stable DNA polymerase

Question 28

STEP 1

DENATURATION:

Answer 28

• The DNA is denatured by heating it to about 95 degrees. This breaks the hydrogen bonds between the base pairs and separates the double-stranded DNA, leaving bases exposed

Question 29

step 2

ANNEALING

Answer 29

• Primer binds to base sequences on either side of DNA (hydrogen bonding)
• The primers are often 20 base pairs long and have complementary bases on either side part of the lenth of DNA
• The primers have different base sequences where on binds to the ‘upward’ strand and the other binds to the ‘downward’ strand
• This temperature is about 60 degrees

Question 30

step 3

EXTENSION:

Answer 30

• The enzyme DNA polymerase uses dNTPs to build new strands of DNA
• The synthesis of new DNA needs 72 degrees
• This is Taq polymerase which was the first heat-stable DNA polymerase and is found in hot springs

Question 31

What are the 2 advantages of Taq polymerase

Answer 31

• Does not get destroyed by the denaturation stage so does not need to be replaced
• It has a high optimum temperature. The temperature in the annaeling process therefore foes not have to be dropped .

Question 32

Define gel electrophoresis

Answer 32

This refers to the seperation of charged molecules such as DNA by differential movement through a gel in an electric field.

Question 33

Describe the most important factors that affect the movement of charged molecules within the gel

Answer 33

• net charge : Negatively charged molecules such as DNA will move towards the anode(+) while negative move towards the cathode(-). High charged molecules move faster than less charged ones
• Size: The smaller the molecules are, the faster they move through the gel
• Composition of the gel:The size of the pores within the gel will determine the speed

Question 34

State the two types of gel and what they are used for

Answer 34

Polyacrlamide gel:
- This is used for seprating small fragments of DNA

Agarose gel:
- This is used for seprating fragments that are between 100 base pairs and 50,000 base pairs

Question 35

Step by step, state the procedure of gel electrophoresis

Answer 35

1.The prep of the gel involves dissolving a powder in hot water. Still fluid is poured into an electrophoresis tank. A ‘comb’ is placed at one end and this results to wells being formed once the gel has solidified
2.When the gel is set, a buffer solution is poured into the tank until it covers the gel in order to give a constant pH
3.A micropipette is used to transfer samples of DNA to all of the wells. The samples of DNA contain a tracking dye in order to track how far they have travelled across the gel
4.A reference sample known as a DNA ‘ladder’ is used to determine the lengths of the fragments of DNA and this can be placed on either ends.
5.A battery pack is connected to the electrodes where the negative electrode is at the same end as the wells, hence allowing the fragments to repel and pull towards the positive electrode.
6.The tracking dye shows how far material in the samples has moved across the gel. when the dye has moved across most of the gel, the battery must be disconnected
7.A stain is added to the gel and is rinsed away to reveal bands across the gel, showing positions of the DNA fragments.

Question 36

Define Microarrays

Answer 36

These are used to identify the genes present in an organisms genome and to find out which genes are expressed within cells.

Question 37

Describe how microarrays analyse genomic DNA

Answer 37

• Probes are used in DNA to identify the known locations across the chromosomes of the organism involved.
• The probes are 500 or more base pairs in length.
• A single microarray can hold probes from the entire human genome.