Application of reproduction & genetics

Question 1

what is the intended purpose of the Human Genome Project & 100K Genome Project?

Answer 1

improve knowledge and understanding of genetic disorders and improve their diagnosis and treatment

Question 2

when did the Human Genome Project begin and end?

Answer 2

1990 & 2003

Question 3

what did the Human Genome Project discover? (give three things)

Answer 3

the order of DNA bases in genomes

the distribution and sequences of introns and exons

the loci of individual genes

Question 4

describe the method of Sanger Sequencing that scientists used which is a method of DNA sequencing

Answer 4

involves reading the base sequence of a length of DNA

sequenced small sections of DNA at a time (usually <1000bps) and took a long time e.g. a year to sequence a million base pairs

Question 5

define genome

Answer 5

all of the genetic information of an organism

Question 6

describe the method of Next Generation Sequencing (NGS)

Answer 6

new rapid technique that sequences an entire genome in a few hours
enable scientists to study variation within the human genome

Question 7

describe the 100k Genome Project

Answer 7

launched in 2012
used NGS to sequence 100,000 genomes of NHS patients with cancer or rare diseases

Question 8

what does the 100K Genome Project enable us to do?
give 5 things

Answer 8

locate genes responsible for rare diseases and cancers

locate mutated genes

compare genomes of genetic disorder sufferers with the normal genome

develop treatments for genetic disorders

undergo effective prenatal diagnosis for genetic disorders

Question 9

what information has the 100K Genome Project provided us?

Answer 9

identifiying allele sequences enables scientists to scan a patient’s DNA sample for mutated sequences and compare the sequence of DNA bases in a patient’s gene to a normal version of the gene

IVF embryos can be screened for the presence of alleles which cause conditions e.g. cystic fibrosis, Huntington’s disease and thalassaemia

genetic screening can be useful in association with genetic counselling, allowing a couple to make informed decisions before having children

Question 10

what could the data produced by the Human Genome Project & 100K Genome Project be used for?

Answer 10

unsure how this information may be used in the future

society has yet to decide how it should be treated and where legal and moral responsibilities lie

Question 11

state a few ethical issues regarding the screening of DNA

Answer 11

owning genetic information could lead to discrimination e.g. health insurance, job applications, social stigmatisation and misuse of data

concerns over routine screening for adult-onset disorders e.g. Alzheimer’s disease and some cancers
some don’t want to learn this info. about themselves, it could cause anxiety

concerns over embryo screening and the potential for choosing alleles to ensure specific characteristics: ‘designer babies’

Question 12

what other species have had their genomes screened and why?

Answer 12

mosquito (anopheles gambiae & plasmodium parasite)
chimpanzees
other primates

allowed scientists to look at revolutionary relationships and to conserve species in the future

Question 13

what are two other methods that could be used to determine how closely related organisms are?

Answer 13

DNA hybridisation
amino acid sequence analysis

Question 14

describe the resistance mosquitos have against insecticide and why is pyrethroid resistance a problem?

Answer 14

rapid evolution of insecticide resistance in the anopheles gambiae mosquito hampers attempts to eradicate the disease responsible for over a million deaths per year

pyrethroid resistance is a problem as it is the only insecticide safe to use on nets that people sleep under

Question 15

when was the DNA sequence for the anopheles gambiae (vector) completed and why is it useful for scientists?

Answer 15

2002
allowing scientists to develop chemicals that could render the mosquito susceptible to insecticides again, preventing it from transmitting malaria

Question 16

describe the resistance and the genome sequencing reasons of the plasmodium sp. parasite

Answer 16

plasmodium sp. has also developed multi-drug resistance

scientists hoped that a better understanding of genetic control of plasmodium infection will allow the development of more effective drugs

Question 17

what does PCR stand for?

Answer 17

polymerase chain reaction

Question 18

what does PCR do and what is it useful for?

Answer 18

PCR amplifies the quantity of DNA rapidly for analysis

a small sample of DNA may be found at a crime scene, but larger samples of DNA are required to carry out tests

using PCR, copies of specific DNA fragments may be made

Question 19

how is variation produced between humans?

Answer 19

exons are regions of DNA that code for proteins

between exons are regions of non-coding DNA called introns which contain blocks of repeated nucleotides

it is the number of times that these blocks are repeated that produce variation

Question 20

what is the name of the repeating blocks of introns found in DNA?

Answer 20

hyper variable regions/short tandem repeats

Question 21

briefly describe how PCR is used to amplify DNA

Answer 21

a number of STR’s used to build up a unique fingerprint

PCR can be used to amplify the STR’s by using a primer which is complementary to the start of the sequence

Question 22

what is a primer?

Answer 22

single stranded DNA
usually 6-25bp in length

Question 23

what are the three stages in PCR?

Answer 23

separation

annealing

extension

Question 24

what happens during (step 1) separation in PCR?

Answer 24

the target DNA molecule is dissolved in a buffer and is heated to 95 degrees

this breaks the hydrogen bonds and denatures the DNA causing the two strands to separate, exposing the nitrogenous bases

Question 25

what happens during (step 2) annealing in PCR?

Answer 25

sample cooled to 50-60 degrees to allow short DNA primers to bind to the DNA strands and form hydrogen bonds with complementary bases

Question 26

what happens during (step 3) extension in PCR?

Answer 26

heating to 70 degrees allows thermally stable DNA polymerase (Taq) to add complementary nucleotides by forming the phosphodiester bonds in the sugar phosphate backbone creating 2 double stranded molecules

Question 27

suggest two practical uses of PCR

Answer 27

forensics - amplification of small quantities of DNA in a blood sample synthesis of a small gene sequence for insertion (by DNA ligase) into a plasmid

Question 28

explain what is meant by the term primer (give two things)

Answer 28

binds by hydrogen bonding to a complementary DNA sequence required for activity of (Taq) DNA polymerase

Question 29

the enzyme ((Taq) DNA polymerase) required at this step is described as thermophilic explain what thermophilic means and why is this important for the process of PCR?

Answer 29

thermophilic: stable to high temp; the enzyme is not denatured at high temp important in PCR as the temperature is increased to 95 degrees and never falls below 55 degrees - this temp. is too high for activity of most enzymes found in nature

Question 30

what are the limitations of PCR? (give 5)

Answer 30

contamination error rate sensitivity to inhibitors limits on amplification DNA fragment size

Question 31

describe the limitation of contamination to PCR

Answer 31

any DNA that enters the system by mistake will be amplified sources may be airborne or come from the previous PCR reaction using the same apparatus

Question 32

describe the limitation of error rate to PCR

Answer 32

Taq polymerase cannot proof-read and correct errors in the base sequence each cycle copies and multiplies DNA , so these errors accumulate

Question 33

describe the limitation of sensitivity to inhibitors to PCR

Answer 33

molecules in the sample may act as inhibitors and PCR is very sensitive to them

Question 34

describe the limitation of limits on amplification to PCR

Answer 34

after about 20 cycles, PCR slows down and plateaus because: reagent concentrations become limiting the enzyme denatures after repeated heating DNA in high concentration causes the single stranded molecules to base pair with each other rather than with primers

Question 35

describe the limitation of DNA fragment size to PCR

Answer 35

PCR is most efficient at making DNA about 1000-3000 base pairs long because taq polymerase can't correct its errors many genes, including human genes, are much longer than this

Question 36

the graph shows the number of DNA molecules made using PCR, starting with one molecule explain the shape of the curve from cycles 1 to 16

Answer 36

doubling (of DNA) after each cycle but very low numbers to start with so appears flat then exponential growth

Question 37

the graph shows the number of DNA molecules made using PCR, starting with one molecule suggest one explanation for the levelling out of the curve from cycles 17 to 20

Answer 37

(suggestion; with explanation) e.g. nucleotides being used up; so there is nothing to make complementary chains with primers are being used up; so they cannot start complementary chains enzymes lose activity/denature; so no polymerisation of complementary strands

Question 38

what is gel electrophoresis?

Answer 38

a method of separating DNA fragments according to size to produce a genetic fingerprint

Question 39

what is the gel in gel electrophoresis made from?

Answer 39

from agarose, similar to agar, which contains pores in its matrix

Question 40

how is gel electrophoresis carried out?

Answer 40

DNA fragments are loaded into wells at one end and a voltage is applied across the gel DNA fragments have a negative charge (on the phosphate group) and so are attracted to the positive electrode smaller fragments will move further because they move through the pores in the gel more easily a DNA ladder can be run alongside the sample; a DNA ladder contains DNA fragments of known length; this can be used to determine the length of DNA in the sample being analysed

Question 41

what are exons and introns?

Answer 41

exons - regions of DNA that code for proteins between exons are regions of non-coding DNA called introns which contain blocks of repeated nucleotides

Question 42

what are the blocks of repeated nucleotides called?

Answer 42

short tandem repeats (STRs) and do not code for proteins

Question 43

what are STRs used for?

Answer 43

to build up a unique fingerprint

Question 44

why do we separate introns instead of exons when producing DNA profiles by gel electrophoresis?

Answer 44

there are far more differences between the introns of individuals than between exons

Question 45

what situations has DNA profiling been used in? there are 6

Answer 45

twins - determining whether babies are monozygotic or fraternal twins siblings - people who have been adopted may wish to determine blood relatives phylogenetic studies to determine relatedness paternity testing immigration forensic use

Question 46

what does genetic engineering allow?

Answer 46

allows DNA to be manipulated, altered or transferred from one form to another

Question 47

describe three scenarios where genetic engineering can be applied

Answer 47

to make useful products - transfer of genes into bacteria e.g. to produce human insulin transfer genes into plants and animals so they acquire new characteristics e.g. disease resistance treating genetic diseases e.g. by transferring genes into humans so they no longer suffer from cystic fibrosis

Question 48

what are the stages for producing a new protein using the DNA technology of a gene transfer? (there are 5)

Answer 48

1- identify and isolate DNA fragments from donor organism using restriction enzymes or reverse transcriptase 2- insert DNA fragments into a vector (recombinant DNA) 3- transfer the recombinant DNA into a suitable host cell 4- identify the host cells which have taken up and are expressing the recombinant DNA 5- clone the host cells

Question 49

what does donor DNA mean?

Answer 49

a DNA fragment containing a desired gene from a donor individual

Question 50

what does vector mean?

Answer 50

used to transfer the donor DNA into the suitable host cell

Question 51

what does recombinant DNA mean?

Answer 51

produced when the donor DNA fragment is spliced into the DNA of another organism

Question 52

what does clone mean?

Answer 52

genetically identical copy

Question 53

what two ways can the desired gene be isolated? (step1-isolating and identifying DNA)

Answer 53

locate it on the donor DNA and cut it out using restriction endonuclease (enzymes that cut DNA as specific base sequences) extract mRNA from a cell actively synthesising the required protein/polypeptide and use reverse transcriptase and DNA polymerase to produce a double stranded cDNA fragment

Question 54

how is reverse transcriptase used (and DNA polymerase) to isolate the DNA?

Answer 54

isolating a gene from within the DNA inside of a nucleus can be difficult however it is likely that there will be large numbers of mRNA molecules in the cytoplasm of a cell that synthesises a lot of this polypeptide reverse transcriptase can be used to produce a single strand of DNA (cDNA) from this mRNA DNA polymerase enzymes can then add free DNA nucleotides to the template strand to produce a double-stranded section of cDNA which codes for the desired protein

Question 55

suggest the source of the reverse transcriptase enzymes

Answer 55

retroviruses (have RNA as their genome)

Question 56

suggest the role of the reverse transcriptase enzymes in the organism you have suggested(?)

Answer 56

DNA can be created from their mRNA this can then be inserted into host cells' DNA for replication

Question 57

what are the five steps to produce cDNA?

Answer 57

1- mRNA is extracted from donor cells e.g. from beta cells in the pancreas (for the gene for insulin production) 2- reverse transcriptase enzymes are used to make a DNA copy of the mRNA 3- the newly synthesised single strand of DNA is called cDNA (complementary DNA) many copies of cDNA are made 4- DNA polymerase enzymes catalyse the addition of free DNA nucleotides this converts the cDNA into a double strand 5- DNA is copied many times using the PCR

Question 58

give 4 benefits of using reverse transcriptase to produce cDNA

Answer 58

overcomes the problem of locating the gene on DNA avoids restriction enzymes cutting the desired gene into non-functional fragments no introns present in cDNA (as these are removed from mRNA) no need for post-transcriptional processing to produce functional mRNA

Question 59

what is a common vector that is used in genetic engineering?

Answer 59

a plasmid these plasmids are isolated from bacteria

Question 60

what is a plasmid?

Answer 60

a small, circular, double-stranded DNA molecule found in bacteria plasmids are separated from chromosomal DNA and can replicate independently

Question 61

what are 4 steps to insert DNA into a vector? (step 2- inserting DNA into a vector)

Answer 61

1- cut the isolated donor DNA using restriction endonuclease (restriction enzymes) these cut the DNA between specific base sequences (restriction sites), such as GAATTC many restriction enzymes cut DNA in a staggered pattern and leave overhanging ends with unpaired bases exposed these are known as 'sticky ends' 2- the same restriction enzymes are then used to cut the plasmid DNA at the same base sequences 3- another group of enzymes, DNA ligase enzymes, are used to splice (join) together the sugar-phosphate backbones of the donor and vector DNA 4- the new plasmid is now known as recombinant DNA

Question 62

why should the same restriction enzyme be used on the DNA isolated for insertion and the vector (plasmid) DNA?

Answer 62

to ensure the unpaired bases on the donor DNA fragment and plasmid are complementary this means complementary bases can form hydrogen bonds

Question 63

describe step 3 of transferring of recombinant DNA into a host cell

Answer 63

these recombinant plasmids are mixed with bacterial cells and calcium chloride in the hope that bacteria will take up the plasmid however, often as few as 1% of the bacterial cells take up the plasmid and become transformed

Question 64

describe step 4 of identifying host cells which have taken up the recombinant DNA and are expressing it

Answer 64

to identify the bacterial cells which have taken up the plasmid and therefore have the desired gene, scientists use a marker gene the marker is usually a gene which confers antibiotic resistance cells which take up the desired gene will also take up the antibiotic resistant gene therefore, if they are grown on a medium containing antibiotics, only the cells containing the plasmids will survive

Question 65

describe step 5 of cloning the host cells

Answer 65

the bacteria can be grown on a large scale to produce large amounts of, for example, insulin

Question 66

give the pros of genetically engineering bacteria

Answer 66

prevention and treatment of disease e.g. produce vaccines allows the manufacture of medical productions e.g. insulin to treat diabetes and human clotting factors to treat haemophilia enhancing crop growth - modified bacteria secrete molecules toxic to pests

Question 67

give the cons of genetically engineering bacteria

Answer 67

plasmids are easily transferred genes may be exchanged with other bacteria and antibiotic resistance marker genes could be transferred to pathogenic bacteria a new microorganism with a new gene may become a threat if released into the environment the possible transfer/activation of oncogenes by using fragments of human DNA

Question 68

what has been a way of increasing food supply for the growing population?

Answer 68

transformed plants which may enhance nutritional value and make crops disease resistant and drought tolerant

Question 69

describe GM soy beans

Answer 69

important source of food used to produce a wide range of products e.g. flour, protein, oil, bread and soya milk 'roundup ready' soybeans are genetically modified to contain genes for herbicide resistance the crops can be sprayed to remove weeds without inhibiting their growth

Question 70

describe Bt tomatoes

Answer 70

Bacillus thuringiensis is a bacterium that lives in the soil it contains genes that produce a protein that acts as an insecticide

Question 71

suggest how the transfer of the bacillus thuringiensis gene into tomato cells would be of benefit?

Answer 71

these genes could be transferred to tomato cells to prevent insects eating the plant; less crop spoilage; higher crop yield

Question 72

describe antisense tomatoes

Answer 72

tomatoes ripen naturally when they produce an enzyme that breaks down their cell wall a second copy of this gene was inserted into tomato plant cells to prevent translation and block the production of this enzyme

Question 73

suggest the benefit of the gene transfer of blocking the enzymes that can break down the cell wall of tomatoes?

Answer 73

less food spoilage during transport longer shelf life

Question 74

give the arguments for GM crops

Answer 74

higher crop yield superior keeping qualities substantial reduction in pesticide use on crops engineered for resistance to fungal pathogens and insect attack

Question 75

give the arguments against GM crops

Answer 75

pollen from GM plants engineered for herbicide resistance might transfer genes to wild relatives/contaminate organic crops reduction in biodiversity such as limited crop varieties and beneficial insects being killed it is claimed that there may be adverse health effects of eating a crop that is expressing a new gene and making a new protein

Question 76

define gene therapy

Answer 76

replacing defective alleles with copies of a new functional DNA sequence

Question 77

how can some genetic diseases be treated?

Answer 77

by replacing genes replicating genes replicating the function of genes using drugs

Question 78

what is the main aim of gene therapy?

Answer 78

to treat a genetic disease by replacing defective alleles in a patient with copies of a new DNA sequence

Question 79

what three methods can be used in gene therapy to introduce DNA into target cells?

Answer 79

a virus as a vector a plasmid as a vector injection of naked plasmid DNA

Question 80

what are two ways of replacing defective genes?

Answer 80

somatic cell therapy germ line therapy

Question 81

what is somatic cell therapy?

Answer 81

targets body cells in affected tissues

Question 82

what are the problems with somatic cell therapy?

Answer 82

effects are usually not permanent (as treated cells die and are replaced) so require repeated treatments not inherited

Question 83

what is germ line therapy?

Answer 83

this is very rare and introduces corrective genes into germ line cells so genetic corrections are inherited

Question 84

suggest why germ line therapy can be controversial?

Answer 84

unpredictable effects, later in life or future generations ethical concerns about genetic modification of future humans currently illegal or highly restricted in many countries

Question 85

describe DMD (Duchenne muscular dystrophy)

Answer 85

it is a recessive, sex-linked form of muscular dystrophy affects up to 1 in 3500 live male births dystrophin is a structural protein found in muscle people with DMD fail to produce dystrophin and consequently have severe muscle loss and often become wheelchair-bound by the time they are teenagers - life expectancy is only 27

Question 86

what are most cases of DMD caused by?

Answer 86

caused by one or more deletions in the dystrophin gene these genes have 79 exons; deletions in any of these alter the reading frame of the dystrophin mRNA the ribosome meets at a stop codon too soon and the full polypeptide is not translated therefore functional dystrophin is not synthesised

Question 87

what is the drug drisapersen and describe it

Answer 87

it is an antisense oligonucleotide it is a sequence of 50 nucleotides that is complementary to the mutated sequence

Question 88

how does the drug drisapersen treat DMD and what is this type of treatment called?

Answer 88

by acting as a 'molecular patch', binding to the mRNA over the exon with the deletion that portion of mRNA becomes double stranded, so the ribosome can't translate it this restores the reading frame, so that a shorter, partially functioning dystrophin protein can be made this type of treatment is called exon skipping

Question 89

how is drisapersen delivered to the patient?

Answer 89

in subcutaneous (under the skin) injections

Question 90

give one advantage and one disadvantage of gene therapy

Answer 90

it has had some success, and the advantages to those who receive it far outweigh the disadvantages however, only a small proportion of the introduced genes are expressed

Question 91

what are some potential issues when using a virus as a vector?

Answer 91

there may be an immune response in the patient the virus may invade non target host cells the virus could potentially become pathogenic and cause disease the virus may affect other genes such as formation of oncogenes

Question 92

what is genomics?

Answer 92

the study of the structure, function, evolution and mapping of genomes as exemplified by the Human Genome Project and the 100K Projects

Question 93

how does the study of genomics improve healthcare?

Answer 93

more accurate diagnosis better prediction of the effect of drugs improved design of drugs new and improved treatments for disease

Question 94

what does the introduction of NGS technology bring about?

Answer 94

it may be possible to look at tailoring therapies to individual patients, where an individual could have a unique treatment for a common disease

Question 95

describe tissue engineering

Answer 95

uses biochemistry, cell biology, engineering and material science to repair, improve or replace biological tissues its goal is to produce 'off the shelf' bio-artificial organs and to regenerate injured tissue in the body

Question 96

describe stem cells

Answer 96

they are undifferentiated cells that have the ability to become many different specialised cell types e.g. meristems stem cells can be used for replacing damaged tissues and organs during tissue engineering when a stem cells divides by mitosis, each daughter cell can remain a stem cell or become another type of cell with a more specialised function

Question 97

where are embryonic stem cells found?

Answer 97

in 3-5 day old embryos

Question 98

where are adult stem cells found and describe them?

Answer 98

in some adult tissues like bone marrow they replace cells that are lost through wear and tear, injury or disease, but they cannot form all cell types

Question 99

list some examples of tissues and/or organs that have been created using tissue engineering

Answer 99

trachea bones bladder skin

Question 100

describe the cells that are grown in tissue culture

Answer 100

they form cell lines that are clones all the cells are derived from a single parent cell and are genetically identical these cell lines can then be used to produce cloned tissue samples or potentially organs

Question 101

give some roles of stem cells in tissue engineering

Answer 101

use to regenerate tissues and organs to screen new drugs to develop model systems to study normal growth and identify the causes of birth defects to investigate the events that occur during human development

Question 102

what are some requirements of using stem cells from embryos?

Answer 102

stem cells left over from IVF are deposited in the UK stem cell bank so they are available for other research groups donors must give specific consent to embryos created with their gametes being used in stem cell research there is no financial reward for any development or discovery made using them

Question 103

does the use of adult stem cells contain ethical issues?

Answer 103

no, they have been used in research and medicine for years

Question 104

how do people justify the use of embryonic stem cells?

Answer 104

they say the use of stem cells helps to clarify biological mechanisms and that a pre-14 day old embryo is a ball of cells with no possibility of independent existence

Question 105

discuss and suggest the possible arguments against the use of stem cells

Answer 105

the source of embryonic stem cells is often from 'spare' embryos left over from IVF an embryo has moral rights but are they to the same degree as a born and living person? fear that the use of stem cells may lead to humans being cloned, an act that fundamentally devalues human life (ethical issues of cloning human tissues and organs)