Manipulating Genomes

Question 1

What is the polymerase chain reaction used for

Answer 1

Generate many copies of DNA fragments - amplification

Question 2

In PCR what is often used to cut DNA fragments

Answer 2

Restriction enzymes

Question 3

What does the reaction mixture for the PCR contain

Answer 3

Original DNA
Primers - small sections of nucleotides
Taq DNA polymerase
And free DNA nucleotides

Question 4

Describe the steps in the PCR (4)

Answer 4

• Double stranded DNA sample
• Heat to 95 degrees - separate strands
• add primers and reduce temp to 55 degreees (allow hydrogen bonds to form) primers anneal (bind)
• raise temperature to 72 degrees DNA polymerase binds and extends primers using free nucleotides

Question 5

How do restriction enzymes work

Answer 5

Used to obtain DNA fragments from a genome

Some sections of DNA have palindromic sequences (read the same both ways - forward and backward)

Restriction enzymes recognise specific sequences and cut/ digest at these sites - cuts can leave sticky ends

Different restriction enzymes cut at different sequences as shape of sequence is complimentary to shape of active site

Question 6

Describe gel electrophoresis

Answer 6

• use of electrical currents to separate DNA/RNA fragments by size
• agarose gel used as it has large pores
• nucleotides are negatively charged so are attracted towards anode
• larger fragments can’t move through agarose as quickly as smaller fragments

Question 7

How are proteins put through gel electrophoresis
- when are they often used

Answer 7

They have different charges so we mix with a chemical first to denature them - they all now have the same charge
- often used in disease diagnosis

Question 8

What is the method for DNA sequencing

Answer 8

Chain termination method
Enables us to determine nucleotide sequences on DNA fragments

Question 9

Why are nucleotides marked with fluorescent dyes

Answer 9

To show what nucleotides are present in a sequence
Mark the end of a fragment - chain terminator

Question 10

Define Pyrosequencing

Answer 10

a nongel-based DNA sequencing technique that detects inorganic pyrophosphate released during DNA synthesis

Question 11

What type of DNA fragments are the chain termination method and pyrosequencing used for

Answer 11

DNA fragments smaller than 1000 base pairs

Question 12

Why do we have to cut genomes into smaller fragments to be able to sequence

Answer 12

Because the chain termination reaction and pyrosequencing can only be carried out with small fragments - less than 1000 base pairs

Question 13

What is BAC

Answer 13

Bacterial artificial chromosome (agent)

an engineered DNA molecule used to clone DNA sequences in bacterial cells

Question 14

What are the uses of sequencing

Answer 14

• allowed for the sequence of amino acids in polypeptide chains to be predicted
• development of synthetic biology - creating biological molecules from scratch
• computational biology ( using a computer to study biology) and bioinformatics ( data store) has also developed
• evolutionary relationships - cytochrome C
• epidemiology ( study of diseases)

Question 15

What does genetic engineering involve

Answer 15

Modifying the genome of an organism by introducing a gene from another organism

Question 16

What do we call organisms that have been genetically engineered

Answer 16

Transgenic or transformed

Question 17

What are the three stages of the genetic engineering process - describe them

Answer 17

1. Use restriction enzymes to isolate the desired gene
2. Recombinant DNA is made - vector DNA is isolated, and cut open using restriction enzyme in step 1. Using same R.E. Means sticky ends are complimentary. DNA fragment inserted into vector DNA using DNA lignase
3. Transforming cells - vector is used to transfer recombinant DNA into bacterial cells (host cells)
   Bacteriophage injects DNA into host cell which integrates with bacterial DNA
   Plasmid vectors are placed in a mixture with host cells and electric field created. This increases permeability of host plasma membranes and allows plasma to enter - electroporation

Question 18

Describe how recombinant DNA is made in genetic engineering

Answer 18

Vector DNA is isolated

Vector DNA is cut open using restriction enzyme that was used to obtain original desired gene

As same R.E. Used, means the sticky ends of vector DNA are complimentary to DNA fragment

DNA fragment inserted into vector DNA using DNA lignase (joins sugar phosphate)

Question 19

Part 3 of genetic engineering process
How are cells transformed

Answer 19

Vector is used to transfer recombinant DNA into bacterial cells (host cells)

Bacteriophage injects DNA into host cell which integrates with bacterial DNA

Plasmid vectors are placed in a mixture with host cells and electric field is created, this increases permeability of host plasma membranes and allows plasmids to enter - electroporation

Question 20

Why have we genetically modified soybeans

Answer 20

They are an important food source but are susceptible to insect pests

GM soybeans contain a gene that codes for a protein that is toxic to some insects

Gene taken from bacteria BACILLUS THURINGIENSIS (B+) and put int plasmid from a second bacteria AGROBACTERIUM TUMEFACIENS

Question 21

What are the two bacterium used to genetically modify soy beans
1 - from where gene is taken
2 - vector DNA

Answer 21

1. Bacillus thuringiensis
2. Agrobacterium tumefaciens

Question 22

What are the ethical issues of genetically modifying crops
Pros and cons

Answer 22

Pros: Less use of chemical pesticides
Increased yield and food

Cons: Monoculture - less biodiversity, vulnerable to disease.

Question 23

What are the pros and cons of genetically modifying pathogens

Answer 23

Pros: Potential treatments of disease
E.g. tumour cells have receptors for poliovirus - GM the virus to inactivate the disease causing genes

Cons: ethical issues -
Lab troubles and mass outbreak of pathogen

If someone with ill intentions gets hold of this knowledge and biowarfare kicks off

Could the GM version um-GM itself and mss outbreak?

Question 24

What is pharming

Answer 24

Genetically modifying animals for drugs

Question 25

What is antithrombin deficiency

Answer 25

Produces too many blood clots

Question 26

How are goats genetically modified to combat antithrombin deficiency

Answer 26

GM a goat embryo and insert it into a female - all offspring produce milk with antithrombin Selective breed desired offspring

Question 27

What are the issues with ownership of GM organisms

Answer 27

Companies will want money for the products Large companies will have a lot of power Poorer countries might not be able to afford the medicines and crops

Question 28

What is gene therapy

Answer 28

Altering alleles inside cells to cure genetic disorders

Question 29

What are the two ways of carrying out gene therapy

Answer 29

1. If the disorder is caused by two mutated recessive alleles, then a working dominant allele can be added 2. If it is caused by a mutated dominant allele then it is ‘silenced’ - fragment of DNA is inserted into the middle of the allele so it no longer works

Question 30

Describe somatic gene therapy What is the problem with this type

Answer 30

Altering somatic cells (diploid) Cystic fibrosis - recessive disease Therefore a functioning dominant allele is inserted into cells lining the lungs Con: offspring would not inherit new gene as sex cells have not been altered

Question 31

What is germline gene therapy

Answer 31

Altering sex cells (haploid), zygotes and early embryonic stem cells (<8days) As a result all cells in organism contain altered DNA so MAY pass this onto offspring Germline therapy in humans is currently illegal

Question 32

What are the ethical debates of gene therapy Pros and cons

Answer 32

Pros: - prolong life of those suffering with life-threatening genetic disorders - improve quality of life as it eases symptoms - germline therapy would allow carriers of genetic disorders to conceive offspring without the disorder - germline therapy could decrease overall number of individuals suffering from genetic disorders and cancers Cons: Technology could be used for other reasons Expensive - could money be going elsewhere Potential the therapy could cause more harm than good

Question 33

What are the disadvantages of gene therapy

Answer 33

- inserted alleles may get overexpressed and result in other problems - vectors could be recognised as foreign and start an immune response - an allele could be inserted into the wrong place within the DNA and lead to unknown impacts - effects may be short-lived in somatic therapy

Question 34

define recombinant DNA

Answer 34

when fragments of DNA are transferred from one organism to another

Question 35

what is DNA sequencing

Answer 35

identifying the base sequence of a DNA fragment

Question 36

examples of benefits of genome-wide comparisons

Answer 36

1. comparing between species - determines evolutionary relationships 2. comparing between individuals of the same species allows us to tailor medical treatment to the individual

Question 37

what is DNA profiling

Answer 37

identifying the unique areas of a person's DNA, in order to create them a profile that is individual to them