CHAPTER 21 - MANIPULATING GENOMES

Question 1

What is a genome

Answer 1

All of the genetic material in an organism

Question 2

What are introns

Answer 2

Large non-coding regions of DNA that are removed from mRNA before it is translated into a polypeptide chain

Question 3

What is satellite DNA

Answer 3

Short sequences of DNA that are repeated many times located within introns/telomeres/centromeres

Question 4

What is the process of producing a DNA profile

Answer 4

Extracting DNA

Digest Sample using restriction endonucleases (different enzymes cut DNA at different places)

Separating DNA fragments - using electrophoresis

Hybridisation - radioactive/fluorescent DNA probes are now added to DNA fragments. DNA probes are short DNA or RNA sequences that are complementary to a known sequence

X-ray images (radioactive labels) or paper/membrane is placed under UV light (fluorescent)

(pg 553)

Question 5

What are some uses of DNA profiling

Answer 5

Paternity tests,
solving crimes - even when a very tiny amount of DNA is available
Risk assessments for diseases

Question 6

What are the steps in Polymerase chain reaction (PCR)

Answer 6

Separating strands - occurs at 90-95 degrees celsius for 30 seconds, denaturing DNA, breaking bonds

Annealing of primers - temp of 55 degrees and primers bond (anneal) to the ends of DNA strands

Synthesis of DNA - temp is increased to 72 degrees for at least a minute, optimum for DNA polymerase to build up the complementary strand, forming 2 new fragments of DNA (and Taq polymerase is used - found in hot springs)

Question 7

State the purpose of the polymerase chain reaction and explain how it has advanced DNA profiling

Answer 7

Polymerase chain reaction (PCR) is a process by which a small piece of DNA is amplified/replicated many times

need relatively large DNA sample for DNA profile/sequencing

in forensic cases/criminal investigations samples available are often extremely small

PCR amplifies samples so DNA profiling can be carried out

Question 8

Discuss the benefits and limitations of DNA profiling

Answer 8

Benefits: can be used in criminal cases to prove guilt or innocence
tiny amounts of DNA can be used
DNA lasts a long time so ‘cold’ cases can be revived by DNA evidence
can be used to prove paternity / to prove or disprove familial relationships in immigration cases
can be used to identify species / can be used to find evolutionary relationships

Limitations: can be too dependent on it and ignore other evidence in criminal cases
DNA profiles can be done at different levels and mistakes can be made
contamination of samples with DNA from other organisms

Question 9

What are the principles for DNA sequencing

Answer 9

DNA is mixed with primer, DNA polymerase and excess of normal nucleotides

Placed in a thermal cycler, intervals of 96 degrees and 50 degrees, separating double stranded DNA into single strands, then added primers to form 2 double stranded

60 degree Celsius DNA polymerase builds up new DNA strand

Each time, a terminator base is incorporated, at different bases, so you can pinpoint each singular base

Order of bases un tubes show sequence of complementary strand of DNA
(pg 559)

Question 10

Produce a flow chart to summarise the main stages of DNA sequencing

Answer 10

DNA for sequencing is mixed with a primer, DNA polymerase, excess of normal nucleotides (bases A, T, C, and G) and terminator bases, each with a coloured fluorescent tag (1);  at optimum temperatures, DNA polymerase builds chains. Whenever a terminator base is incorporated, the chain is terminated and no more bases can be added (1);  process is repeated until all possible chains are created (1);  chains separated out by gel electrophoresis; (1)  lasers used to read fluorescent tags and learn order of bases in complementary DNA strand, from this original strand can be deciphered

Question 11

What is the difference in the time it takes to sequence the genetic material of a bacterium today compared to the first complete bacterial genome in 1995, and explain the reasons for the difference in times

Answer 11

Original bacterial genome – around 548 days, high-throughput methods – under 1 day. Difference is 547 days

In original techniques each stage was carried out by hand in the laboratory

modern techniques mainly carried out in machines, many DNA fragments processed at once, so much faster and more efficient

Question 12

What are terminator bases

Answer 12

Bases that when added to a DNA chain during DNA synthesis terminate it / no more nucleotides can be added

Question 13

Why are terminator base is so important in both the sangha method of sequencing, and in the more modern high-throughput sequencing methods

Answer 13

Using terminator bases all possible length DNA fragments are synthesised

having different coloured fluorescent tags attached to four different terminator bases

makes it possible to work out sequence of original DNA

once chains have been separated using gel electrophoresis

high- throughput sequencing is much more complex and rapid

but still relies on terminator bases to terminate chains in final stages

Question 14

What is bioinformatics

Answer 14

The development of the software and computing tools needed to organise and analyse raw biological data

Question 15

What is computational biology?

Answer 15

Computers using the data obtained from bioinformatics to build theoretical models of biological systems, which can be used to predict what will happen in different circumstances

Question 16

What has the sequencing of Genomes of pathogens enabled doctors to do

Answer 16

Find out the source of infection

Identify antibiotic resistant strains of bacteria

Track progress of an outbreak of a potentially serious disease

Identify regions in the gene of pathogens that may be useful targets in the development of new drugs

Question 17

What is DNA barcoding

Answer 17

Identifying particular sections of the genome that are common to all species but vary between them, so comparisons can be made -

Question 18

In animals and plants , what section is chosen for DNA barcoding

Answer 18

649 base-pair section of Mitochondrial DNA in the gene cytochrome c oxidase, coding for an enzyme in respiration

For plants, DNA in 2 regions of chloroplasts are selected

Question 19

What is proteomics

Answer 19

Study and amino acid sequencing of an organisms, entire protein complement

Question 20

What are exons

Answer 20

regions of DNA that are transcribed to RNA and retained after introns are spliced out

Question 21

What do spliceosomes do

Answer 21

mRNA needs to be modified, so introns are removed, and some of the exons

Exons can then be joined together buy enzyme complexes - spliceosomes, to give the mature functional mRNA

Question 22

What is synthetic biology

Answer 22

The design and construction of novel artificial biological pathways organisms, or devices, or the redesign of existing natural biological systems

Question 23

What are the techniques involved in synthetic biology?

Answer 23

Genetic engineering

Use of biological systems or parts of them in industrial contacts e.g. mobilise enzymes

Synthesis of new genes to replace faulty genes eg. cystic fibrosis

Synthesis of an entire new organism

Question 24

Explain the impact of computational, biology and bioinformatics on the usefulness of DNA sequencing to scientists

Answer 24

Bioinformatics allows scientists to analyse large amounts of data generated during sequencing of billions of base pairs in genomes ]

display the data in ways that make sense and help identify patterns

Computational biology takes these results and uses them to build up models

e.g., of the spread of disease, the evolutionary relationships between organisms, the inheritance of antibiotic resistance in bacteria

and use them to model possibilities in different circumstances

Question 25

Explain how the ability to sequence the gene and can be used to identify the source of an outbreak of infectious disease and how this is helpful

Answer 25

ach strain of a pathogen has a slightly different genome and so can be accurately identified by DNA sequencing

ability to identify a particular strain of a pathogen means country/place of origin can be identified (for treatment, quarantine, etc.)

or individuals with disease can be identified (for isolation, treatment, etc.)

spread of a strain of disease can be tracked and transmission methods understood to prevent further spread

Question 26

Discuss how DNA sequencing has change the ways in which we identify species and our understanding of evolutionary relationships

Answer 26

Traditionally species identification was done by observation of anatomical and physiological features

with DNA sequencing genome similarities are examined and comparisons made to standard species genome

much more accurate but harder in the field

in understanding evolutionary relationships DNA sequencing looks at the difference in number of mutations between species

and by calculating average mutation rate

you can calculate when two species diverged

Question 27

One gene one polypeptide is an outdated concept. Discuss how our model of the link between the genotype and phenotype is changing.

Answer 27

Human genome sequencing shows there are 20–25 000 coding genes (1);

proteomics suggests there are between 250 000 to 1 000 000 proteins (1);

technology used to sequence DNA can also sequence amino acids in proteins – this is not always what you would predict from bases in DNA

some genes can code for up to 1000 different proteins

introns and some exons may be removed before mRNA lines up on ribosomes

spliceosomes combine exons in different orders to give different versions of mRNA

which code for different amino acid sequences and different proteins

some proteins are modified by other proteins once they are formed

may remain intact, shortened or lengthened which creates other proteins

Question 28

What is genetic engineering

Answer 28

Genetic engineering is the practical technique of isolating genes for desirable characteristics in one organism

and placing them into another organism

using a suitable vector

Question 29

Explain the difference between the way restriction in the nucleus is reversed. Transcriptase is produce genes ready for insertion into another organism.

Answer 29

Restriction endonucleases cut a required gene from DNA of an organism at specific sites within DNA molecule

may leave uneven ends and extended regions of unpaired bases are called sticky ends

used for inserting into DNA of a vector

Reverse transcriptase enzymes create DNA from mRNA used to make a particular protein

reverse of normal DNA transcription

synthesised DNA usually inserted into a vector same way as method using restriction endonucleases

Question 30

Describe how a gene is inserted into a bacterial plasmid, which is then taken up by the bacteria and how scientist ensure that they can identify bacteria that have been successfully transformed

Answer 30

Plasmid cut open using same restriction endonucleases as used to excise the gene to be used

so sticky ends of DNA fragment/gene and cut plasmid are complementary/match

sticky ends lined up and DNA ligase joins them by forming phosphodiester bonds between the two strands of DNA

transgenic plasmids taken up by bacteria as a result of calcium ions and temperature making membrane permeable to plasmid or by electroporation when electric current makes membrane porous to plasmids

plasmids have gene for antibiotic resistance, so bacteria that take up engineered plasmids can be identified

plasmid also contains marker gene engineered in (site where plasmid is cut open) to show engineering has worked – usually fluorescence or enzyme which changes colour of medium – required gene is inserted in middle of marker – so bacteria showing marker trait are not successfully engineered

Question 31

produce a Flow diagram to show the process of genetic engineering, a bacterium

Answer 31

infection of plant cell with bacteria containing engineered plasmid

callus formation

and growing on of cloned cells to form many individual transgenic plants

production of functional transgenic bacterium after plasmid is reabsorbed into host bacterial cell

Question 32

What are ethical concerns of genetic manipulation of microorganisms

Answer 32

Inserting human genes into microorganisms

Question 33

What are the pros and cons of (GM) pest resistance organisms

Answer 33

Pros - Reduce amount of pesticide spraying, protects environment, helping poor farmers, increases yield

Cons - non-pest insects and insect eating predators might be damaged by the toxins of GM plants, insects may become resistant to pesticides in GM crops

Question 34

What are the pros and cons of (GM) disease resistance organisms

Answer 34

Pros - increases yield

Cons - transferred genes may spread to wild populations and cause problems eg. superweeds

Question 35

What are the pros and cons of (GM) herbicide resistance organisms

Answer 35

Pros - reduces competition of weeds, hence increasing yield

Cons - biodiversity could be reduced if herbicides are overused to destroy weeds, fear of superweeds

Question 36

What are the pros and cons of (GM) extended shelf-life on organisms

Answer 36

Pros - Extended shelf life hence reducing food waste

Cons - Reduces the commercial value and demand for the crop

Question 37

What are the pros and cons of (GM) growing conditions for organisms

Answer 37

Pros - crops can grow in a wider range of conditions eg. flood resistance or drought resistance

Cons - …….

Question 38

What are the pros and cons of (GM) nutritional value to organisms

Answer 38

Pros - nutritional value of crops can be increased eg. enhanced vitamins

Cons - Allergy to proteins made in GM crops

Question 39

What are the pros and cons of (GM) medical uses of GM organisms

Answer 39

Pros - Plants can be used to produce human medicines and vaccines

Cons - …….

Question 40

What are some examples of GM animals

Answer 40

Swine fever-resistant pigs
Faster-growing salmon

Question 41

What is pharming

Answer 41

Genetic engineering animals to produce human medicines

Question 42

What are the two aspects to pharming

Answer 42

Creating animal models - adding or removing genes so animals develop certain diseases, acting as models for new therapies (test subjects)

Creating human proteins - human gene coding for a medically required protein (which bacteria may not be able to do), and can produce milk with desired protein in

Question 43

What are the ethical issues with pharming

Answer 43

Should animals be genetically engineered to act as models for human disease?
is it right to put human genes into animals?
Is it acceptable to put genes from with another species into an animal without being certain it won’t harm them?
Does GE’ing them reduce them to commodities
Is welfare compromised?

Question 44

What diseases can be replaced by gene therapy and how

Answer 44

CF, Haemophillia, SCIDS as thy are a result of faulty (or mutant) genes

Different methods can be used to remove alleles and replace with healthy ones

Question 45

What is the downside for somatic cell gene therapy

Answer 45

Only a temporary solution for a treated individual

Question 46

What are some issues, which until recently, have been seen in somatic gene therapy

Answer 46

Getting healthy alleles into the affected cells

Getting the engineered plasmids into the nucleus of the cells

Starting and maintaining expression of the healthy allele

(using viral vectors)

Question 47

What is germ line gene therapy

Answer 47

Inserting a healthy allele into the germ cells, usually the eggs or embryo immediately after fertilisation, so the individual would be born healthy with the normal allele in place, and would pass it on to their own offspring

Question 48

What are some ethical issues with germ line cell gene therapy

Answer 48

Potential impact on an individual of an intervention on the germ cells is unknown,

Human rights of an unborn individual could be said to be violated - done without consent, and is irreversible

Tech may enable designer babies

Question 49

Suggest why there is relatively little debate about the ethics of genetically engineering micro organisms

Answer 49

Genetically engineered microorganisms have been used safely for many years

genetically engineered microorganisms produce many beneficial materials from insulin to antibiotics so benefits are very tangible

little empathy for microorganisms and no welfare issues

Question 50

Potential benefits of genetically engineering plans

Answer 50

pest-resistant
amount of pesticide spraying needed (1); protects other insects in the environment and helps poor G

farmers who cannot afford to spray regularly (1); yield can be increased giving more food (1); weed killer resistant soya beans allow farmers to spray to get rid of weeds without affecting crop which then gets all the resources and so has bigger yield (1); other plants are engineered specifically for high yield (1); crops can grow in wider range of conditions/survive adverse conditions e.g., scuba rice developed by IRRI to help rice farmers cope with extreme weather events (1); extended shelf-life of some GM crops reduces food waste – either less food has to be produced or there is more food to go around (1); nutritional value of crops can be increased – enhanced levels of vitamin A in golden rice, enhanced protein, or carbohydrate in increased yields (1); plants used to grow medicines – vaccines in bananas, tomatoes, etc., antibiotics (1); disease resistant varieties can be reduced which reduces crop losses and provides more food

Question 51

Discuss the ethical issues with genetically engineering plants

Answer 51

Non-pest insects might be damaged by toxins in GM plants – for example Bt protein in modified soya plants could affect larvae of other moths and butterflies

antibiotic genes could spread from marker genes into wild populations and spread antibiotic resistance

transferred genes might mutate

biodiversity could be reduced if GM crops are herbicide resistant

people might be allergic to proteins grown in GM crops, e.g., Bt protein

insects may become resistant to pesticides in plant tissues. Two or more insecticide genes may be used to reduce chances of resistance developing

Question 52

Describe the process of genetically modifying animals to produce human proteins and discuss some of the ethical issues this raises

Answer 52

Process:
Copy of human gene coding for relevant/desired protein is isolated or synthesised, introduced into genetic material of fertilised cow, sheep, or goat egg
promoter sequence added to ensure gene is expressed only in mammary glands fertilised, (female) transgenic embryo is returned to mother to grow to birth. When mature transgenic animal conceives and gives birth, it produces milk containing desired human protein to be harvested
well-being of transgenic animals appears unaffected – simply make an extra protein in their milk
enormous benefits for human patients receiving transgenic proteins/pharmaceuticals,

Ethical issues:
mother animal has to undergo fertility treatment to produce eggs and then embryos are placed in surrogate animals, which have to be medically-prepared
success rate of inserting human gene is low, so ova/embryos are wasted and destroyed
process involves germ line cell manipulation so genes are passed on in perpetuity