Module 6 Section 4: Manipulating Genomes

Question 1

What are the different techniques needed to study genes

Answer 1

Cutting out DNA fragments using restriction enzymes
Polymerase chain reaction (PCR)
DNA sequencing
Gel electrophoresis

Question 2

How is PCR used

Answer 2

Can be used to select a fragment of DNA (containing the gene or area of DNA you’re interested in) and amplify it to produce millions of copies
Occurs in a few hours

Question 3

Full process of PCR

Answer 3

A reaction mixture is set up that contains the DNA sample, free nucleotides, primers and DNA polymerase.
The DNA mixture is heated to 95 °C to break the hydrogen bonds between the two strands of DNA.
DNA polymerase doesn’t denature even at this high temperature
The mixture is then cooled to between
50 and 65 °C so that the primers can bind (anneal) to the strands.
The reaction mixture is heated to 72 °C so DNA polymerase can work.
The DNA polymerase lines up free DNA nucleotides alongside each template strand.
Complementary base pairing means new complementary strands are formed.
Two new copies of the fragment of DNA are formed and one cycle of PCR is complete.
The cycle starts again, with the mixture being heated to 95 °C and this time all four strands (two original and two new) are used as templates

Question 4

How are primers and DNA polymerase used in PCR

Answer 4

Primers are short pieces of DNA that are complementary to the bases at the start of the fragment you want.
DNA polymerase is an enzyme that creates new DNA strands.

Question 5

Why is it important that DNA polymerase doesn’t denature at the temperatures PCR uses

Answer 5

It means many cycles of PCR can be carried out without having to use new enzymes each time

Question 6

How much DNA can a PCR cycle produce

Answer 6

Each PCR cycle doubles the amount of DNA,
e.g. 1st cycle = 2 × 2 = 4 DNA fragments, 2nd cycle = 4 × 2 = 8 DNA fragments, 3rd cycle = 8 × 2 = 16 DNA fragments

Question 7

What is electrophoresis used for

Answer 7

Uses an electrical current to separate out DNA fragments, RNA fragments or proteins depending on their size

Question 8

How to set up apparatus for electrophoresis

Answer 8

Pour agarose gel into a gel tray and leave to solidify.
A row of wells is created at one end of the gel.
Put the gel tray into a gel box (or tank).
Make sure end of the gel tray with the wells is closest to the negative electrode on the gel box.
Add buffer solution to the reservoirs at the sides of the gel box so that the surface of the gel becomes covered in the buffer solution.

Question 9

How to load DNA samples into wells

Answer 9

Use micropipette to add the same volume of loading dye to DNA fragments (from PCR or restriction enzymes)
Loading dye helps samples sink to the bottom of the wells to make them easier to see
Add 10μl of DNA sample to first well
Make sure tip of micropipette is in buffer solution and just above opening of the well
Don’t stick tip too far into well as it may pierce bottom
Repeat process and add same volume of the other DNA samples to other wells in the gel
Clean tip of micropipette each time
Record the DNA added to each well

Question 10

How to carry out electrophoresis

Answer 10

Put lid on box and connect leads from gel box to power supply
Pass 100V through the gel
Run gel for 30mins or until dye is 2cm from end of the gel
Remove gel tray from the gel box and tip off excess buffer solution
Stain DNA fragments by covering surface of the gel with staining solution then rinsing gel with water
Bands of the different DNA fragments will now be visible

Question 11

How to measure the size of the DNA fragments

Answer 11

Size of DNA fragment is measured in bases
E.g. ATCC = 4 bases or base pairs
1000 bases is one kilobase (1 kb)

Question 12

How do DNA fragments separate in electrophoresis

Answer 12

DNA fragments are negatively charged so they’ll move through the gel towards positive electrode at far end of the gel (anode)
Small DNA fragments move faster and travel further through gel
Means DNA fragments separate according to size

Question 13

How are RNA fragments separated using electrophoresis

Answer 13

This follows the same basic method as for DNA fragments

Question 14

How are proteins separated using electrophoresis

Answer 14

Proteins can be positively charged or negatively charged
Before they undergo electrophoresis, they’re mixed with a chemical that denatures the proteins so they all have the same charge.

Question 15

Uses of electrophoresis of proteins

Answer 15

e.g. to identify the proteins present in urine or blood samples, which may help to diagnose disease.

Question 16

What are palindromic sequences of nucleotides

Answer 16

These sequences consist of the same order of bases when read backwards on the opposite strand

Question 17

What are restriction enzymes

Answer 17

Restriction enzymes are enzymes that recognise specific palindromic sequences (known as recognition sequences) and cut (digest) the DNA at these places
They cut the DNA via hydrolysis reactions
Also called restriction endonucleases

Question 18

How can you choose what type of DNA fragment are formed

Answer 18

Can choose between different restriction enzymes
They can cut at different specific recognition sequences, because the shape of the recognition sequence is complementary to an enzyme’s active site.
Results in different fragments forming
E.g. the restriction enzyme EcoRI cuts at GAATTC, but HindIll cuts at AAGCTT.

Question 19

How to use a restriction enzyme in a lab

Answer 19

The DNA sample is incubated with the specific restriction enzyme

Question 20

What do you need in order to completely separate a fragment from the whole chain

Answer 20

If recognition sequences are present at either side of the DNA fragment you want, you can use restriction enzymes to separate it from the rest of the DNA.

Question 21

How are restriction enzymes used to bind different fragments together

Answer 21

Sometimes the cut leaves sticky ends
These are small tails of unpaired bases at each end of the fragment.
Sticky ends can be used to bind (anneal) the DNA fragment to another piece of DNA that has sticky ends with complementary sequences.

Question 22

What is a DNA profile

Answer 22

Some of an organism’s genome consists of repetitive, non-coding base sequences which are called short tandem repeats and are within satellite DNA
These are sequences that don’t code for proteins and repeat over and over
E.g. introns, telomeres (caps and tails), centromeres
The number of repetitions of non-coding sequences differs from person to person, so the length of these sequences in nucleotides differs too.
This is their DNA profile

Question 23

PCR is used to increase DNA sample size

Answer 23

The number of times a short tandem repeat is repeated at different, specific places (loci) in a person’s genome (and so the number of nucleotides there) can be analysed using electrophoresis.
This creates a DNA profile.

Question 24

Where is DNA profiling useful

Answer 24

PCR and DNA profiling is performed on traces of DNA left at the crime scene
eg. blood, semen, skin cells, saliva or hair
DNA profile is compared to a sample taken from a suspect or identified using criminal data base

Can be used to prove paternity of a child

Can be used in immigration cases to prove or disprove family relationships

DNA profiling is used to identify which species an organism belongs to
Used to demonstrate evolutionary relationships between different species

Can be used to identify individuals who are at risk of developing particular diseases
Certain non-coding microsatellites, or the repeating patterns they make, have been found to be associated with an increased risk or particular disease E.g. Huntington’s disease, sickle cell anaemia

Question 25

Process of DNA sequencing

Answer 25

DNA for sequencing is mixed with a primer, DNA polymerase, a excess of normal nucleotides (containing bases A, T, C, G) and terminator bases Mixture undergoes PCR in thermal cycler At 72°C DNA polymerase starts to build up new DNA strands by adding nucleotides with the complementary base to the single-strand DNA template Each time a terminator base is incorporated instead of a normal nucleotide, the synthesis of DNA is terminated since no more bases can be added As the chain-terminating bases are present in lower amount and are added at random, this results in many DNA fragments of different lengths depending on where the terminating bases have been added All possible DNA chains will be produced after many cycles as the reaction is stopped at every base Fragments separated according to their length by capillary sequencing which is like gel electrophoresis in small capillary tubes Final base is identified on each strand by fluorescent markers Lasers detect different colours which shows the order of the sequence The order of bases in capillary tubes shows the sequence of the complementary strand of DNA which is used to determine the sequence of the original DNA strand

Question 26

How whole genomes be studied quickly

Answer 26

Chain termination technique still used but the tube contains all the modified nucleotides each with a fluorescent label The machine reads the sequence out High throughput sequencing can read 1000 times more bases in a given time

Question 27

How does high throughput pyrosequencing work

Answer 27

Section of DNA cut into fragments, split into single strands and a strand from each fragment is attached to a small bead PCR used to amplify fragments on each bead Each bead put into separate well Free nucleotides added to the wells attach to the DNA strands via complementary base pairing Wells also contain specific enzymes which emit light when bases are added to DNA strand Computers analyse occurrence and intensities of the light emitted in the different wells and process the information to interpret the DNA sequence Technique can sequence about 400 million bases in a 10 hour period

Question 28

Overview of process of how chain termination method is used to study whole genomes

Answer 28

Genome fragmented Fragments inserted into BACs DNA extracted from colony DNA sequenced using chain termination method Fragments put back in order

Question 29

First step of preparing genome to be sequenced: BACs

Answer 29

Chain termination technique can only sequence fragments up to 750bp long Genome is cut into smaller fragments (about 100 000 bp) using restriction enzymes. Fragments are inserted into bacterial artificial chromosomes (BACS) The BACs are then inserted into bacteria Each bacterium contains a BAC with a different DNA fragment

Question 30

Second step of preparing genome to be sequenced: DNA extracted from bacteria

Answer 30

The bacteria divide to create colonies of cloned (identical) cells that all contain a specific DNA fragment. Together the different colonies make a complete genomic DNA library. DNA is extracted from each colony and cut up using restriction enzymes (to make fragments small enough to sequence) Produces overlapping pieces of DNA.

Question 31

Third step of sequencing whole genomes: fragments sequenced

Answer 31

Each piece of DNA is extracted from BAC and sequenced using the chain-termination method (including terminator base types of all nucleotides) Fragments separated out according to size with electrophoresis, fragments differ by 1 base each Electrophoresis sheet then put through computer where laser reads the bases (by radioactive tag of fluorescent marker) and creates base sequence Finally the DNA fragments from all the BACs are read out by computers, to complete the entire genome.

Question 32

What are bacterial artificial chromosomes

Answer 32

These are man-made plasmids Fragments are inserted into these BACs which are inserted into bacteria to create colonies of different fragments Not needed specifically to sequence genome Just there to provide genomic library so the fragments are to hand if needed

Question 33

What can you deduce from sequencing a gene

Answer 33

The sequence of amino acids the gene codes for Used to find primary structure of polypeptide

Question 34

What is synthetic biology

Answer 34

This is how biological molecules are made through predicting the structure of a protein derived from a gene that has been sequenced

Question 35

Uses of synthetic biology

Answer 35

Building biological systems from artificially made molecules (e.g. proteins) Can see whether they work in the way we think they do. Redesigning biological systems to perform better and include new molecules. Designing new biological systems and molecules that don't exist in the natural world, but could be useful to humans, e.g. energy products (fuels) and drug products. Can also be used to build artificial pathways, organisms or devices

Question 36

How is synthetic biology different from genetic engineering

Answer 36

Genetic engineering involves the direct transfer of DNA trom one organism to another Whereas in synthetic biology DNA is created from scratch

Question 37

How is artemisinin a product of synthetic biology

Answer 37

Artemisinin is an antimalarial drug Used to be obtained by extracting it from a plant. Using synthetic biology, scientists have created all the genes responsible for producing a precursor to artemisinin. Successfully inserted these genes into yeast cells We can now use yeast to help produce artemisinin

Question 38

How are gene sequences and whole genome sequences compared and what can they be compared between

Answer 38

Can be compared between organisms of different species and organisms of the same species Involves computational biology and bioinformatics

Question 39

What is computational biology

Answer 39

Using computers to study biology and analyse large amounts of biodata Can build theoretical models of biological systems to predict what will happen in different circumstances Can work out 3D structures of proteins and understanding processes such as gene regulation e.g. to create computer simulations and mathematical models

Question 40

What is bioinformatics

Answer 40

Developing and using computer software that can analyse, organise and store biological data in very large quantities

Question 41

What areas of biology involves the comparison of gene sequences and genomes

Answer 41

To study genotype-phenotype relationships Epidemiological studies To help understand evolutionary relationships

Question 42

How does studying genotype-phenotype relationships use comparison of gene sequences and genomes

Answer 42

Used to predict a phenotype by analysing the genotype Using DNA sequencing we now know that there are many more unique proteins than coding genes in human DNA This suggests that the sequence of amino acids (phenotype) is not always what would be predicted from the genome sequence alone

Question 43

Example of using the comparison of gene sequences and genomes to study genotype-phenotype relationships: Marfan syndrome

Answer 43

Marfan syndrome is a genetic disorder caused by a mutation of the FBN1 gene Position and nature of mutation on gene affects the symptoms the person has (vision, cardiovascular system or muscles) Sequencing FBN1 gene of sufferers and documented them along with phenotypes Bioinformatics allowed scientists to compare all the data and identify genotype-phenotype correlations Can help with treatment as gene sequencing can help predict what problems they may face

Question 44

How do epidemiological studies use comparison of gene sequences and genomes

Answer 44

Epidemiological is the study of health and disease within a population Considers distribution, causes and effects of disease Gene mutations have been linked to a greater risk of disease Computerised comparisons between genomes of people with a disease and those without can be used to detect particular mutations that could be responsible for the increased risk of disease However, the risk of developing diseases has been found to be a product of the environment and our genes

Question 45

How can the comparison of gene sequences and genomes be used to understand evolutionary relationships

Answer 45

Whole genomes of different species can be sequenced They are then analysed using computer software to see how closely related different species are All organisms evolved from shared common ancestors Closely related species evolved away from eachother more recently so share more common DNA

Question 46

How can the comparison of gene sequences and genomes be used to understand human migratory patterns

Answer 46

When groups of humans separated and moved to different parts of the world their genomes changed in slightly different ways Can use computers to compare the genomes of people from different parts of the world Can be used to build picture of early human migration

Question 47

4 steps or genetic engineering

Answer 47

1. Isolate the gene 2. Package the gene (in a vector) 3. Transfer the vector to the recipient cell 4. Recipient cell expresses the gene

Question 48

Processes of isolating the gene in genetic engineering

Answer 48

Use DNA probe to identify the gene then use restriction enzymes to cut the gene out The restriction enzymes used are specific to DNA strands which have a certain base sequence DNA fragments are left with sticky ends Harvest mRNA from cells where it is being expressed Use reverse transcriptase to convert mRNA to cDNA (complementary DNA) Add primers and polymerase to build double stranded DNA

Question 49

Processes packaging gene into a vector in genetic engineering

Answer 49

Gene can be sealed into bacterial plasmids This uses same restriction enzymes to leave the complementary sticky ends on bacterial DNA Once fragment sticky ends are lined up with bacterial sticky ends, DNA ligase forms phosphodiester bonds between the sugar and the phosphate groups on the two strands of DNA (joins up sugar phosphate backbones), this is called ligation Attenuated viruses or yeast chromosomes also used Recombinant DNA is now formed

Question 50

Processes transferring the vector to the recipient cell in genetic engineering

Answer 50

Multiple methods of transformation Plasmids taken up by host cell Can use electroporation where a high voltage pulse to disrupt the membrane and allow plasmids to move into cells Can also culture bacterial cells and plasmids in a calcium rich solution and increase temperature which causes bacterial membrane to become permeable and plasmids can enter Bacteriophage vector infects the bacterium by injecting its DNA into it The phage DNA with desired gene in it then integrates into the bacterial DNA

Question 51

What do vectors do in genetic engineering and what is commonly used

Answer 51

Most commonly used are bacterial plasmids Plasmids can recombine with host DNA to form recombinant DNA

Question 52

How do scientists identify if transformation has taken place in genetic engineering

Answer 52

Plasmids that are used as vectors usually have a marker gene May have a gene for antibiotic resistance which enables scientists to determine that the bacteria have taken up the plasmid by growing the bacteria in a media containing antibiotic After recombinant DNA is formed the vector plasmids are given second marker gene to show plasmid contains recombinant gene If DNA fragment is inserted successfully the marker gene will not function indicating process has worked Marker genes can be for antibiotic resistant, fluorescence or enzyme which causes colour change

Question 53

Current uses for genetically modified microorganisms

Answer 53

Microorganisms can be genetically modified to produce substances that are useful to people such as insulin or vaccines which can be produced in large quantities this way GM microorganisms are used ti store living record of DNA of another organism in DNA libraries

Question 54

Future uses of genetically modified microorganisms

Answer 54

GM pathogens can be used in research for cancer treatment Scientists found tumour cells have receptors on their membranes for the poliovirus So the poliovirus will recognise and attack them By genetically engineering polioviruses to inactivate genes that cause poliomyelitis, scientists can use it to kill cancer cells without causing disease This can lead a treatment for cancer

Question 55

Positive ethics for use of genetically modified microorganisms

Answer 55

This means that previously untreatable diseases can now be treated which reduces the suffering they cause

Question 56

Negative ethics for use of genetically modified microorganisms

Answer 56

Scientists researching pathogens could become infected with the live pathogen and cause an outbreak of disease Genetically modified version of pathogen could revert back to its original form and cause an outbreak of disease Knowledge of how to genetically engineer dangerous pathogens could be used to create agents for biowarfare Researchers have to follow strict protocols to prevent these

Question 57

Current uses for genetically modified plants

Answer 57

Scientists inserted a gene originally found in bacteria which codes for the Bt protein The Bt protein is toxic to some of the insects that feed on soybeans Soy beans can also be engineered to be resistant to a common weed killer and to contain Bt protein Means that farmers can spray to get rid of weeds and reduce the use of pesticides Leads to much higher yield of soya beans

Question 58

Positive ethics of use of genetically modified plants

Answer 58

Can reduce amount of chemical pesticides farmers use on crops which harm the environment Increases availability of food for famine prone countries

Question 59

Negative ethics of use of genetically modified plants

Answer 59

GM soybean plants can encourage monoculture which decreases biodiversity and could leave the whole crop vulnerable to disease because all plants are genetically identical Growing genetically modified crops can mean that the normal crop is not grown anymore, if a new pest is introduced that kills all the modified variants, a possible gene that could make them resistant to that pest may be present in the normal crop which has been lost Transferred genes may spread to wild populations to produce super weeds

Question 60

Future uses of genetically modified plants

Answer 60

Can be used to grow crops which are resistant to pesticides Fruits can be genetically modified to contain various vaccines which can stop the spread of disease for a fraction of the normal price Crops can be modified to grow in hostile environments such as salt water

Question 61

Applications of genetically modified animals

Answer 61

Swine fever resistant pigs Faster growing salmon

Question 62

What is pharming

Answer 62

Genetically engineering animals to create human medicines or pharmaceuticals

Question 63

Types of uses of pharming

Answer 63

Creating animal models for certain diseases so they can act as models for new therapies Creating human proteins by introducing human gene coding for a medically required proteins

Question 64

Example of pharming

Answer 64

Hereditary anti thrombin deficiency is a disorder that makes blood clots more likely to form in the body The risk of forming blood clots can be reduced with infusions of the protein antithrombin Scientists found a way to produce high yields of this protein using goats DNA fragments that code for human antithrombin are injected into goat embryo Embryo implanted into female goat Offspring is tested for presence of protein when it’s born If it does it is selectively bred to produce a herd of goats that produce antithrombin in their milk Protein is extracted from milk and used to produce the drug ATryn that can help treat people with the deficiency

Question 65

Positives of pharming

Answer 65

Drugs can be made in large quantities to make them more available to people

Question 66

Negatives of pharming

Answer 66

Manipulating animals genes can cause harmful side effects for the animal It enforces the idea that animals are merely assets that can be treated however we choose

Question 67

How can genetically engineered organisms be owned by companies

Answer 67

Scientists from different institutions collaborate knowledge to improve techniques of genetic engineering A group of scientists or the company they work for may want to obtain a patent to control, by law, who uses the product and how for a set period of time

Question 68

Positive ethical issues of getting a patent for a GM organism

Answer 68

Owner of patent will get money generated from selling the product Encourages scientists to compete to be the first to come up with a new beneficial genetic engineering idea Means we get products faster

Question 69

Negative ethical issues of getting a patent for a GM organism

Answer 69

Farmers in poorer countries can’t afford patented genetically modified seeds If they can afford it for one year, the patent means they can’t use it for more than one year without paying again

Question 70

What happens in gene therapy

Answer 70

Involves altering alleles inside cells to cure genetic disorders

Question 71

Why may there be two ways to cure a genetic disorder using gene therapy

Answer 71

What approach is used depends on whether the disorder is caused by a dominant or recessive allele

Question 72

How to carry out gene therapy to cure a disorder caused by two recessive alleles

Answer 72

You can add a working dominant allele to make up for them

Question 73

How to carry out gene therapy to cure a disorder caused by a dominant allele

Answer 73

You can 'silence' the dominant allele e.g. by sticking a section of DNA in the middle of the allele so it doesn't work any more

Question 74

How to get new allele inside of cell in gene therapy

Answer 74

Allele inserted into cells using vectors These can be: altered viruses, plasmids or liposomes (spheres made of lipid)

Question 75

Types of gene therapy

Answer 75

Somatic therapy Germ line therapy

Question 76

What is somatic gene therapy

Answer 76

Involves altering the alleles in body cells Such as cells that are most affected by the disorder have the mutant allele replaced by healthy allele E.g. cystic fibrosis (CF) is a genetic disorder that's damaging to respiratory system Somatic therapy for CF targets the epithelial cells lining the lungs. Somatic therapy doesn't affect sex cells so any offspring could inherit the disease Only temporary solution as healthy cells are replaced by stem cells which have faulty allele through mitosis

Question 77

What is germ line therapy

Answer 77

Involves altering the alleles in the sex cells. Means that every cell of any offspring produced from these cells will be affected by gene therapy and they won't inherit the disease. Germ line therapy in humans is currently illegal though.

Question 78

Positive ethical issues of gene therapy

Answer 78

Prolong lives of people with genetic disorders Improve quality of life for people with genetic disorders Carriers of genetic disorders can conceive a baby without disorder or risk of cancer (only germ line) Decrease number of people suffering from genetic disorders (germ line)

Question 79

Negative ethical issues of gene therapy

Answer 79

Technology can be used in ways other than medical treatment like treating effects of aging Potential of doing more harm than good by using technology E.g. risk of overexpression of genes Gene therapy is expensive and health services should spend resources on treatments which have passed clinical trials instead

Question 80

Disadvantages of gene therapy

Answer 80

Effects may be short lived (somatic only) Patient may have to undergo multiple treatments (somatic only) May be difficult to get allele into specific body cells Body could identify vectors as foreign bodies and start immune response against them An allele could be inserted into the wrong place in DNA, possibly causing cancer An inserted allele can be overexpressed leading to too much of the missing protein

Question 81

How is DNA profiling used in forensic science

Answer 81

Samples of DNA from crime scene is taken and isolated E.g. semen, skin cells, saliva, hair PCR used to amplify areas containing short tandem repeats (repeats in DNA unique to individuals) Primers used to bind to either side of repeats so whole repeat amplified PCR products run on electrophoresis gel and DNA profiles produced are compared to see if any match samples collected from suspects Match by having same pattern on bands on gel If samples match it links a person to a crime scene

Question 82

How can DNA profiling be used in medical diagnosis

Answer 82

Can be used to analyse risks of genetic disorders Useful when the specific mutation isn’t known Useful when several mutations could have caused disorder as it identifies a broader, altered gene pattern

Question 83

Example of how DNA profiling is used in medical diagnosis

Answer 83

Preimplantation genetic haplotyping (PGH) screens embryos created by IVF for genetic disorders before they're implanted into the uterus. The faulty regions of the parents' DNA are used to produce DNA profiles These are compared to the DNA profile of the embryo. If the profiles match, the embryo has inherited the disorder. Can be used to screen for cystic fibrosis or Huntington's disease etc

Question 84

How to collect results from gel electrophoresis

Answer 84

Hybridisation: DNA probes are added to label the fragments These radioactive probes attach to specific fragments Development: Membrane with radioactively labelled DNA fragments is placed onto an X-ray film Development of the X-ray film reveals dark bands where the radioactive or fluorescent DNA probes have attached

Question 85

Process of producing a DNA sample

Answer 85

Extracting DNA using reverse transcriptase on mRNA or DNA probe (small pieces amplified by PCR) Digesting sample using restriction endonucleases Separation using electrophoresis to produce clear recognisable pattern Hybridisation using radioactive or fluorescent probes (p Analysing evidence

Question 86

Why would a genome have to be fragmented before sequencing

Answer 86

Takes less time Smaller fragments to sequence improves accuracy You can divide the job over different labs to save time

Question 87

Difference between RNA polymerase and DNA polymerase

Answer 87

RNA polymerase: Makes mRNA, tRNA or rRNA by transcription Uses one strand of DNA and forms one strand DNA polymerase: Uses in DNA replication Semi conservative replication where both strands are used and 2 strands are formed Used before cell division