The Control of Gene Expression (3.8) Flashcards
1
Q
Mutations (AO1)
Define gene mutation.
A
Random change in the base sequence of DNA.
Results in the formation of new alleles.
2
Q
Mutations (AO1)
Gene mutations might occur spontaneously during _____________.
A
DNA replication
3
Q
Mutations (AO1)
List the types of mutations
A
Addition of bases;
Deletion of bases;
Substitution of a base,
Inversion of bases,
Duplication of bases / genes
Translocation of bases / genes;
4
Q
Mutations (AO1)
A change in the DNA base sequence may alter the [1] structure, this may alter the [2] of amino acids.
This may change the [3] of hydrogen, ionic and disulphide bonds between the [4] groups of amino acids.
This may change the [5] structure of the polypeptide.
A
[1] primary
[2] sequence
[3] position
[4] R
[5] tertiary
5
Q
Mutations (AO1)
The mutation rate is __________ by mutagenic agents.
A
increased
6
Q
Mutations (AO1)
List examples of mutagenic agents
A
High energy radiation e.g. x rays, gamma rays, ultraviolet light
Carcinogens e.g. benzene
Biological agents e.g. viruses
7
Q
Mutations (AO1)
A __________ mutation may change only one triplet code.
A
substitution
8
Q
Mutations (AO1)
Due to the ______________ nature of the genetic code, not all mutations result in a change to the encoded amino acid.
A
degenerate
9
Q
Mutations (AO1)
3 key terms for the genetic code
A
degenerate
universal
non-overlapping
10
Q
Mutations (AO1)
What is meant by the degenerate nature of the genetic code?
A
More than one triplet / codon codes for a single amino acid
11
Q
Mutations (AO1)
What is meant by the universal nature of the genetic code?
A
Same 3 bases used in DNA (triplets) / mRNA (codon) code for the same amino acids in all organisms
12
Q
Mutations (AO1)
What is meant by the non-overlapping genetic code?
A
Each base is read only once in the triplet / codon
13
Q
Mutations (AO1)
Additions and [1] of bases may change the nature of all base [2] downstream from the mutation.
This results in a [3] shift.
A
[1] deletions
[2] triplets
[3] frame
14
Q
Mutations (AO1)
A base substitution may result in the formation of a [1] codon.
This signals to the [2] to detach from mRNA and polypeptide during translation.
This results in a [3] polypeptide.
A
[1] stop
[2] ribosome
[3] shorter
15
Q
Mutations (AO1)
What happens during a translocation mutation?
A
Sections of DNA bases relocate from one area of the genome to another;
e.g., between non-homologous pairs of chromosomes;
16
Q
Mutations (AO1)
A
Box 2
Inversions do not change the number of DNA bases
17
Q
Mutations (AO2)
Sickle cell disease (SCD) is a group of inherited disorders. People with SCD have sickle-shaped red blood cells. A single base substitution mutation can cause one type of SCD. This mutation causes a change in the structure of the beta polypeptide chains in haemoglobin. Explain how (3 marks).
A
1. Change in primary structure OR sequence of amino acids;
2. Change in (position) of hydrogen / ionic / disulfide bonds;
3. Alters tertiary structure;
18
Q
Stem Cells (AO1)
Cells become specialised through the process of cellular ______________.
A
differentiation
19
Q
Stem Cells (AO1)
Functions of stem cells
A
Differentiation;
Mitosis;
20
Q
Stem Cells (AO1)
4 types of stem cells
A
Totipotent
Pluripotent
Multipotent
Unipotent
21
Q
Stem Cells (AO1)
________________ cells occur only for a limited time in early mammalian embryos.
A
Totipotent
22
Q
Stem Cells (AO1)
Totipotent cells can divide by [1] and produce [2] type of body cell.
A
[1] mitosis
[2] ANY
23
Q
Stem Cells (AO1)
TRUE or FALSE:
During development, totipotent cells translate only part of their DNA, resulting in cell specialisation.
A
TRUE
24
Q
Stem Cells (AO1)
Pluripotent cells are found in ___________
A
embryos
25
# **Stem Cells (AO1)**
Functions of pluripotent stem cells
**1.** Divide in unlimited numbers;
**2.** Produce MOST cell types
**3.** Used to treat human disorders
26
# **Stem Cells (AO1)**
Which stem cells
are found in mature mammals?
multipotent
unipotent
27
# **Stem Cells (AO1)**
Multipotent stem cells produce ________ cell types
SOME
28
# **Stem Cells (AO1)**
Unipotent stem cells in the heart can differentiate into ___________
cardiomyocytes
29
# **Stem Cells (AO2)**
Myelodysplastic syndromes (MDS) are a group of malignant cancers. In MDS, the bone marrow does not produce healthy blood cells.
Haematopoietic stem cell transplantation (HSCT) is one treatment for MDS. In HSCT, the patient receives stem cells from the bone marrow of a person who does not have MDS. Before the treatment starts, the patient’s faulty bone marrow is destroyed.
For some patients, HSCT is an effective treatment for MDS. Explain how (*3 marks*).
**1.** Stem cells differentiate/produce healthy (blood) cells;
**2.** No MDS/faulty/cancerous (blood) cells;
**3.** Stem cells divide/replicate by mitosis;
30
# **Stem Cells (AO2)**
Scientists have investigated the use of different types of stem cell to treat damage to the heart after a myocardial infarction. During a myocardial infarction, a number of different cell types in the heart die. This includes cardiomyocytes which are heart-muscle cells.
Embryonic pluripotent stem cells (ESCs) can divide and differentiate into a wide range of different cell types.
Using the information given, suggest one reason why ESCs might be suitable to treat damage to the heart.
(ESCs produce MOST types of cell)
So they can replace any type of heart cell;
31
# **Stem Cells (AO2)**
Embryonic pluripotent stem cells (ESCs) have not yet been used to treat people who have had a myocardial infarction. This is because of concern that the use of ESCs might lead to more harm to the person.
Suggest how putting ESCs into a person’s heart might lead to more harm to the person (*3 marks*).
**1.** Differentiating into the wrong types of cells.
**2.** Might divide out of control;
**3.** Leading to tumour / cancer;
32
# **Stem Cells (AO2)**
Haematopoietic stem cell transplantation (HSCT) is a long-term treatment for sickle cell disease (SCD). In HSCT, the patient receives stem cells from the bone marrow of a person who does not have SCD.
The donor is often the patient’s brother or sister. Explain why.
Less chance of rejection
by immune system
(from brother/sister);
33
# **Stem Cells (AO1)**
What are iPS cells?
induced pluripotent stem cells
34
# **Stem Cells (AO1)**
Induced pluripotent stem (iPS) cells can be produced from adult [1] cells using appropriate 'reprogramming' protein [2] factors.
[1] **somatic**
(e.g. keratinocytes in the skin)
[2] **transcription**
35
# **Stem Cells (AO1)**
Suggest how transcription factors can reprogramme cells to form iPS cells (*2 marks*).
**1.** Bind to DNA promoter region;
**2.** Stimulate / inhibit RNA polymerase
**3.** Increase / decrease transcription
36
# **Stem Cells (AO1)**
Advantages of using iPS cells
**1.** Somatic cells easy to obtain;
**2.** Divide in unlimited numbers;
**3.** Produce MOST cell types;
**4.** Used to treat human disorders via transplants;
**5.** Less chance of rejection by immune system
(as using somatic cells that originated from patient);
37
# **Stem Cells (AO1)**
Disadvantages of using iPS cells?
**1.** Takes a long time to differentiate into desired specialised cell;
**2.** Cells generated 'in vitro' (i.e. in cell culture) may not function when transplated back into humans;
38
# **Regulation of Transcription & Translation (AO1)**
What binds to the DNA promoter region?
Transcription Factors
| These are proteins with specific tertiary structures
39
# **Regulation of Transcription & Translation (AO1)**
The promoter region is located BEFORE or WITHIN the gene?
Before
| Sometimes this is referred to as 'upstream' of the gene
40
# **Regulation of Transcription & Translation (AO1)**
Transcription factors are found in the [1] and upon activation can enter the [2].
[1] cytoplasm
[2] nucleus
41
# **Regulation of Transcription & Translation (AO1)**
What happens after the transcription factor binds to the promoter region?
**stimulates RNA polymerase**;
transcription begins and mRNA increases;
42
# **Regulation of Transcription & Translation (AO1)**
**TRUE or FALSE:**
Transcription factors binding to the promoter regions **always** increase transcription.
**FALSE**
| Some transcription factors inhibit transcription
43
# **Regulation of Transcription & Translation (AO1)**
How does oestrogen enter cells?
**1.** Lipid-soluble (steroid hormone);
**2.** Diffuses through the phospholipid bilayer;
44
# **Regulation of Transcription & Translation (AO1)**
What does oestrogen bind to?
oestrogen receptor (ER alpha)
| The receptor is a transcription factor
45
# **Regulation of Transcription & Translation (AO1)**
What happens to the oestrogen receptor (ER alpha) upon binding with oestrogen?
Changes its tertiary structure;
| The receptor now acts as a transcripton factor
46
# **Regulation of Transcription & Translation (AO1)**
**TRUE or FALSE:**
Oestrogen is a transcription factor
FALSE
| Its receptor when activated by oestrogen binding = transcription factor
47
# **Regulation of Transcription & Translation (AO1)**
Osterogen binds to its receptor becuase it has a _____________ shape
complementary
48
# **Regulation of Transcription & Translation (AO1)**
The activiated ooestrogen receptor (ER alpha) is a [1] and binds to the [2] region.
This stimulates [3] and leads to transcription of a gene.
[1] transcription factor
[2] promoter
[3] RNA polymerase
49
# **Regulation of Transcription & Translation (AO2)**
Steroid hormones are hydrophobic.
Explain why steroid hormones can rapidly enter a cell by passing through its cell-surface membrane [*2 marks*].
**1.** Lipid soluble;
**2.** Diffuse through phospholipid bilayer;
50
# **Regulation of Transcription & Translation (AO2)**
In the cytoplasm, testosterone binds to a specific androgen receptor (AR).
An AR is a protein.
Suggest and explain why testosterone binds to a specific AR (*2 marks*).
**1.** Testosterone has a specific tertiary structure;
**2.** This has a complementary shape to the receptor;
| Many hormones are 'modified' proteins
51
# **Regulation of Transcription & Translation (AO2)**
The binding of testosterone to the androgen receptor (AR) changes the shape of the AR.
This AR molecule now enters the nucleus and
stimulates gene expression.
Suggest how the AR could stimulate gene expression (*2 marks*).
**1.** AR is a transcription factor;
**2.** Binds to DNA promoter region;
**3.** Stimulates RNA polymerase;
52
# **Regulation of Transcription & Translation (AO1)**
RNA interference (RNAi) inhibits the ________________ of mRNA
translation
53
# **Regulation of Transcription & Translation (AO1)**
Types of RNA interfering molecules
small interfering RNA (siRNA)
micro RNA (miRNA)
54
# **Regulation of Transcription & Translation (AO1)**
Are siRNA and miRNAs single **OR** double stranded?
single stranded
55
# **Regulation of Transcription & Translation (AO1)**
siRNA binds to specific [1] molecules via [2] base pairing.
This guides enzymes which [3] mRNA.
[4] is prevented.
[1] mRNA
[2] complementary
[3] destory
[4] translation
56
# **Regulation of Transcription & Translation (AO1)**
If a specific protein is still produced in smaller quantities this could be because not all mRNA has been __________________ by RNA interference.
destroyed
57
# **Regulation of Transcription & Translation (AO2)**
**1.** siRNA binds to mRNA for CENP-W;
(via complementary base pairing)
**2** (mRNA for CENP-W) destroyed
**3.** Prevents translation of CENP-W;
**4.** As CENP-W reduces so does tubulin production;
58
# **Regulation of Transcription & Translation (AO1)**
Define epigenetics
**Inheritable** changes in gene function;
without changes to the DNA base sequence;
59
# **Regulation of Transcription & Translation (AO1)**
Types of epigenetic changes
Methylation
Acetylation
60
# **Regulation of Transcription & Translation (AO1)**
**TRUE or FALSE:**
Changes in the environment lead to changes in methylation and acetylation which determine whether a gene is expressed.
TRUE
61
# **Regulation of Transcription & Translation (AO1)**
During methylation, a methyl group is added to a ______ _______.
DNA base
| typically cytosine or guanine in the promoter region
62
# **Regulation of Transcription & Translation (AO1)**
Methylation of DNA bases in the [1] region, prevents transcription [2] from binding.
This [3] transcription.
[1] promoter
[2] factors
[3] decreases / inhibits
63
# **Regulation of Transcription & Translation (AO1)**
Chromosomal DNA in eukaryotes is wrapped around _____________
histones
64
# **Regulation of Transcription & Translation (AO1)**
Acetylation of histone makes them [1] packed.
This makes the promoter region and target gene more accessible to transcription factors and [2].
This [3] transcription.
[1] loosely
[2] RNA polymerase
[3] increases
65
# **Regulation of Transcription & Translation (AO1)**
Decreasing methylation, ________________ transcription
stimulates / increases
66
# **Regulation of Transcription & Translation (AO1)**
Explain why decreasing acetylation of histones decreases transcription.
**1.** Histones are more tightly packed;
**2.** Prevents transcription factors from binding to the promoter region;
**3.** Prevents RNA polymerase from accessing the target gene;
67
# **Regulation of Transcription & Translation (AO1)**
68
# **Gene expression and cancer (AO1)**
Cancer is caused by [1] mitosis and this can lead to a abnormal mass of cells known as a [2].
[1] uncontrolled
[2] tumour
69
# **Gene expression and cancer (AO1)**
Benign tumour characteristics
slow growing;
surrounded by a capsule;
do NOT metastasise;
70
# **Gene expression and cancer (AO1)**
Malignant tumour characteristics
fast growing;
non-capsulated;
metastasise;
(spread to other parts of body)
71
# **Gene expression and cancer (AO1)**
The rate of tumour development is controlled by which genes?
Tumour suppressor genes
Oncogenes
72
# **Gene expression and cancer (AO1)**
Role of tumour suppressor genes
Slow down / regulate the rate of cell cycle
73
# **Gene expression and cancer (AO1)**
Role of oncogenes
Speed up the rate of cell cycle;
Leads to uncontrolled cell cycle / mitosis
74
# **Gene expression and cancer (AO1)**
How do oncogenes arise?
Random mutations in proto-oncogenes
| Proto-oncogenes are normal genes which speed up the cell cycle
75
# **Gene expression and cancer (AO2)**
BRCA1 and BRCA2 are human genes that code for tumour suppressor proteins.
Mutations in BRCA1 and BRCA2 can cause cancer. Explain how (*3 marks*).
**1.** Change in DNA base sequence;
**2.** Change in primary structure / sequence of amino acids
OR Change in tertiary structure
**3.** Results in rapid / uncontrollable cell division / mitosis;
76
# **Gene expression and cancer (AO1)**
Increasing methylation of which gene could lead to the development of a tumour?
Tumour suppressor genes
| As increasing methylation decreases transcription
77
# **Gene expression and cancer (AO1)**
Increasing acetylation of histones near which gene could lead to the development of a tumour?
Oncogenes
78
# **Gene expression and cancer (AO2)**
Treatment with drugs might be able to reverse the epigenetic changes that cause cancers.
Suggest and explain how (*3 marks*).
**1.** Increase methylation of oncogene(s);
**2.** Increasing methylation inhibits / decreases transcription;
**3.** Decrease methylation of tumour suppressor gene;
**4.** Decreasing methylation stimulates / increases transcription;
**5.** Increase acetylation of histones stimulates transcription / gene expression;
(e.g. of tumour suppressor gene)
79
# **Gene expression and cancer (AO2)**
Myelodysplastic syndromes (MDS) are a group of malignant cancers of blood cells.
MDS can develop from epigenetic changes to tumour suppressor genes. In some patients, the drug AZA has reduced the effects of MDS. AZA is an inhibitor of DNA methyltransferases. These enzymes add methyl groups to cytosine bases.
Suggest and explain how AZA can reduce the effects of MDS in some patients (*3 marks*).
**1.** AZA reduces methylation of DNA/cytosine;
**2.** Tumour suppressor gene is transcribed/expressed;
Accept mRNA produced for transcription/transcribed.
**3.** Prevents rapid/uncontrollable mitosis
OR cell division can be controlled/stopped/slowed;
80
# **Gene expression and cancer (AO2)**
Increased methylation of the promoter region of a tumour suppressor gene causes one type of human throat cancer.
In this type of throat cancer, cancer cells are able to pass on the increased methylation to daughter cells. The methylation is caused by an enzyme called DNMT.
Scientists have found that a chemical in green tea, called EGCG, is a competitive inhibitor of DNMT. EGCG enables daughter cells to produce messenger RNA (mRNA) from the tumour suppressor gene.
Suggest how EGCG allows the production of mRNA in daughter cells (*3 marks*).
**1.** EGCG binds to active site of DNMT;
**2.** DNMT cannot methylate / less methylation of promoter region of tumour suppressor gene;
**3**. Transcription factor(s) can bind to promoter region;
**4.** RNA polymerase stimulated;
81
# **Gene expression and cancer (AO1)**
Increased concentration of which hormome are linked to the development of some breast cancers?
Oestrogen
82
# **Gene expression and cancer (AO2)**
ER-positive breast cancers have receptors for the hormone oestrogen. These cancers develop as a result of increased oestrogen concentrations in the blood. Effective treatment of ER-positive breast cancers often involves the use of drugs which have a similar structure to oestrogen.
Suggest and explain how these drugs are an effective treatment of ER-positive breast cancers (*3 marks*).
**1.** Drug binds to oestrogen/ER receptor;
**2.** Prevents binding of oestrogen
**3.** No/fewer transcription factor(s) bind to promoter
OR
RNA polymerase not stimulated
83
# **Genome projects (AO1)**
Define genome
all the DNA in a cell
| Some viruses have a RNA genome
84
# **Genome projects (AO1)**
____________ sequencing has allowed entire genomes of multiple organisms to be fully sequenced.
Automated
85
# **Genome projects (AO1)**
To sequence a genome means to know the exact sequence of ___________ that make up the entire DNA of an organism.
bases
86
# **Genome projects (AO1)**
List source(s) / location(s) of genomes
- Linear DNA (arranged as chromosomes) in eukaryotes
- Circular DNA in prokaryotes, chloroplasts, mitochondria
- Plasmids in bacteria
- Viral DNA or RNA (e.g. HIV)
87
# **Genome projects (AO1)**
If researchers know which genes are present in a genome, they can determine which ______________ can be produced via gene expression.
proteins
88
# **Genome projects (AO1)**
**TRUE or FALSE:** only 1% of the human genome contains genes that code for proteins
TRUE
89
# **Genome projects (AO1)**
Define proteome
the full range of proteins produced by cells
90
# **Genome projects (AO1)**
Applications of the proteome
The identification of potential antigens for use in vaccine production
91
# **Genome projects (AO1)**
Knowledge of the genome has led to increased study of non-coding DNA and regulatory DNA such as.......
- promoters
- terminators
- enhancers
- siRNAs
- miRNAs
- tRNAs
92
# **Genome projects (AO1)**
**TRUE or FALSE:**
Non-coding DNA is located in intergenic* DNA sequences and makes up most of the DNA.
| *Intergenic means in between genes
TRUE
93
# **Genome projects (AO1)**
Determining the genome of the viruses could allow scientists to develop a vaccine.
Explain how (*2 marks*).
**1.** Could identify the proteome;
**2.** Then identify potential antigens
(to use in the vaccine);
94
# **Genome projects (AO1)**
Name **two techniques** the scientists may use when analysing viral DNA to determine whether two viruses are closely related.
- DNA/genome sequencing;
- The polymerase chain reaction;
- Genetic/DNA fingerprinting;
- Gel electrophoresis;
95
# **Recombinant DNA technology (AO1)**
What is recombinant DNA technology?
The transfer of fragments of DNA from one organism / species, to another.
| e.g. Bacteria with plasmids that continue a human gene
96
# **Recombinant DNA technology (AO1)**
Give two reasons why bacteria are able to use human DNA to produce human proteins (*2 marks*).
**1.** The genetic code is **universal**
**2.** The mechanism of transcription is **universal**;
**3.** The mechanism of translation is **universal**;
97
# **Recombinant DNA technology (AO1)**
List the 5 steps in recombinant DNA technology in the correct order
**1. Isolation** of DNA (usually contains a gene)
**2. Insertion** of DNA into a vector (e.g. a plasmid)
**3. Transformation** of cells (to produce a genetically modfied or transgenic organism with two or more sources of DNA).
**4. Identification** of cells that have taken up the DNA by using marker genes
**5. Growth / cloning**
- i.e. bacterial divide by binary fission
- amplify DNA using PCR
98
# **Recombinant DNA technology (AO1)**
3 ways to isolate a DNA fragment
which typically contains a gene
**1.** Reverse transcriptase & mRNA
**2.** Restriction endonucleases
**3.** Gene machine
99
# **Recombinant DNA technology (AO1)**
Describe how isolated mRNA from a cell can be converted into DNA (*3 marks*).
**1.** mRNA is mixed with free DNA nucleotides AND **reverse transcriptase**.
**2.** Free DNA nucleotides bind to single stranded mRNA template via complementary base pairing.
**3.** **Reverse transcriptase joins DNA nucleotides together** to form a single stranded cDNA molecule.
**4.** DNA polymerase is required to make cDNA double stranded.
| cDNA means copy DNA i.e. it is a copy based on mRNA
100
# **Recombinant DNA technology (AO1)**
Advantages of using reverse transcriptase to isolate DNA fragments
- Introns have been removed
- Cells producing protein will contain many mRNA molecules
- mRNA is easy to isolate from cells
101
# **Recombinant DNA technology (AO1)**
Disadvantages of using reverse transcriptase to isolate DNA fragments
- Many steps involving involving enzyme-controlled reactions
- Time consuming
- Requires more technical expertise
102
# **Recombinant DNA technology (AO1)**
Restriction endonucleases are enzymes that [1] phosphodiester bonds at specific DNA base sequences called [2] sites.
1. hydrolyse
2. restriction
103
# **Recombinant DNA technology (AO1)**
Restriction sites are often _______________
palindromic
104
# **Recombinant DNA technology (AO1)**
What is the consequence if the restriction site for the restriction endonuclease occurs within the DNA fragment researchers wish to isolate.
This will cut the gene and it will not code for a functional protein.
105
# **Recombinant DNA technology (AO1)**
What type of ends are produced after cutting DNA with restriction endonucleases.
Sticky ends
Blunt ends
106
# **Recombinant DNA technology (AO1)**
What are sticky ends used for?
To insert a gene into a vector
(e.g. a plasmid)
107
# **Recombinant DNA technology (AO1)**
What are blunt ends used for?
Can be amplified by the polymerase chain reaction;
Separated by size using gel electrophoresis;
108
# **Recombinant DNA technology (AO1)**
Advantages of using restriction endonucleases to isolate DNA fragments
- Produce sticky and blunt ends
- 1000s of restriction endonucleases have been isolated that are each highly specific to different DNA sequences
109
# **Recombinant DNA technology (AO1)**
Disadvantages of using restriction endonucleases to isolate DNA fragments
**Contains introns**
Enzymes may cut in the middle of the desired gene leading to a non-functional protein
110
# **Recombinant DNA technology (AO1)**
Restriction endonucleases
111
# **Recombinant DNA technology (AO1)**
Which method of DNA isolation is decribed below:
*Desired sequence of nucleotide bases are entered into a computer and automated machinery synthesises the DNA fragment*
Gene machine
112
# **Recombinant DNA technology (AO1)**
Advantages of using gene machines to isolate DNA fragments
- Faster process owing to automated machinery and fewer enzyme controlled reactions
- Sequences contain no introns
- Blunt and sticky ends can be added
113
# **Recombinant DNA technology (AO1)**
Disdvantages of using gene machines to isolate DNA fragments
If sequence of DNA is unknown, requires the primary structure of the polypeptide to be known.
114
# **Recombinant DNA technology (AO1)**
Once a DNA fragment has been isolated, what must be added to enable gene expression?
- A promoter region
(allow transcription factors to bind)
- A terminator region
(ensures only the DNA fragment is transcribed)
115
# **Recombinant DNA technology (AO1)**
Suggest and explain one reason why bacteria might not be able to produce every human protein (*1 mark*).
Cannot splice pre-mRNA, so cannot remove introns
OR
Do not have Golgi apparatus, so cannot process/modify proteins;
OR
Do not have the required transcriptional factors, so cannot carry out transcription/produce mRNA;
116
# **Recombinant DNA technology (AO1)**
A geneticist concluded it would be faster to create a human gene using a gene machine than by using reverse transcriptase to convert mRNA into cDNA.
Suggest why the geneticist reached this conclusion.
**Faster** to use gene machine than all the enzyme-catalysed reactions (involving reverse transcriptase);
117
# **Recombinant DNA technology (AO1)**
M = promoter;
N = terminator;
118
# **Recombinant DNA technology (AO1)**
Which methods of DNA isolation could used prior to insertion of the human gene into a bacteria to ensure the protein could be produced.
**1.** mRNA & reverse transcriptase AND gene machine
**2.** Produce DNA / human gene without introns;
**3.** Bacteria cannot remove introns / cannot splice mRNA / cannot splice pre-mRNA;
119
# **Recombinant DNA technology (AO1)**
Define a vector
A DNA carrier that can be used to transfer foreign DNA into cells
120
# **Recombinant DNA technology (AO1)**
Examples of vectors
Bacterial plasmid;
Viruses
121
# **Recombinant DNA technology (AO1)**
Describe how enzymes could be used to **insert** a human gene into a plasmid (*2 marks*).
**1.** Cut the plasmids with the **same** restriction endonuclease (used to isolate the gene);
**2.** Both have **complementary sticky ends**;
**3.** (Mix together) and add **DNA ligase** to join human gene to the plasmid vector (via phosphodiester bonds);
122
# **Recombinant DNA technology (AO1)**
What bond forms between the sticky ends?
Hydrogen bonds
| via complementary base pairing
123
# **Recombinant DNA technology (AO1)**
What bond does DNA ligase form to join the isolated DNA to the plasmid?
Phosphodiester
124
# **Recombinant DNA technology (AO1)**
Describe how a bacterial cell be transformed?
Heat or electric shock;
Creates short-lived pores in the cell surface membrane;
Allows plasmids (with DNA of interest) to be taken up;
125
# AS Recap (Cells)
Describe binary fission in bacteria
(3 marks).
**1.** Replication of circular DNA;
**2.** Replication of plasmids;
**3.** Division of cytoplasm
(to produce daughter cells);
126
# **Recombinant DNA technology (AO1)**
Why are not all cells transformed?
**1)** Not all the vectors take up DNA to become recombinant
*(unsuccessful insertion)*
**2)** Not all the cells take up recombinant vectors
*(unsuccessful transformation)*.
127
# **Recombinant DNA technology (AO1)**
What type of gene is need to identify if a cell has successfully been transformed / is transgenic / genetically modified?
Marker gene
128
# **Recombinant DNA technology (AO1)**
Define a marker gene
Allow easy identification of cells that have taken up the vector (e.g. plasmid) with the DNA fragment / gene of interest
129
# **Recombinant DNA technology (AO1)**
3 examples of marker genes
1. Green flourescent protein (GFP)
2. Antibiotic resistance
3. Lactase (which produces a blue product)
130
# **Recombinant DNA technology (AO1)**
What happens if a gene of interest is inserted into the middle of the ampicillin resistance gene in a plasmid?
Damages the gene;
Codes for non-functional protein;
Bacteria will be destroyed if explosed to amplicillin
(no longer resistant to antibiotic);
131
# **Recombinant DNA technology (AO1)**
To enable researchers to identify which bacteria have taken up the plasmid with a human gene, the plasmids contain a gene that codes for a green fluorescent protein (GFP).
Bacteria that contain this plasmid glow green under UV light.
Suggest one advantage of using this gene for GFP to identify bacteria that have taken up plasmids (*1 mark*).
Can quickly identify transformed bacteria using UV light;
132
# **Recombinant DNA technology (AO1)**
What does PCR stand for?
Polymerase chain reaction
133
# **Recombinant DNA technology (AO1)**
The polymerase chain reaction is used to ____________ .
amplify DNA
134
# **Recombinant DNA technology (AO1)**
What temperature is required to break the hydrogen bonds in DNA during PCR?
95oC
135
# **Recombinant DNA technology (AO1)**
What is a primer?
Short single stranded DNA molecule;
Has complementary bases to the start of the DNA fragment;
Extended by DNA polymerase;
136
# **Recombinant DNA technology (AO1)**
What temperature is required for the 'annealing'* of primers to single stranded DNA?
| *annealing is primers binding to each end of the template strands
55oC
137
# **Recombinant DNA technology (AO1)**
What type of DNA polymerase is required for PCR?
thermostable
138
# **Recombinant DNA technology (AO1)**
What temperature is used during the synthesis stage of PCR?
72oC
139
# **Recombinant DNA technology (AO1)**
**TRUE or FALSE:**
Thermostable DNA polymerases denatures at 72oC?
FALSE
140
# **Recombinant DNA technology (AO1)**
Describe how an isolated gene can be replicated by the polymerase chain reaction (PCR) (*4 marks*).
**1.** Heat DNA to 95oC to break (weak) hydrogen bonds / separate strands
**2.** Add primers and add DNA nucleotides
**3.** Cool to 55oC to allow ‘annealing’ of primers and DNA nucleotides via complementary base pairing
**4.** Add thermostable DNA polymerase
(e.g. Taq polymerase);
**5.** Heat to 72oC
**6.** DNA polymerase joins nucleotides together by phosphodiester bonds to synthesise new strand by extending the primers;
141
# **Recombinant DNA technology (Maths)**
Which formula can be used to calculate the number of DNA strands produced after PCR?
142
# **Recombinant DNA technology (Maths)**
Assuming we started with one DNA molecule, how many DNA molecules are produced after 25 PCR cycles?
143
# **Recombinant DNA technology (Maths)**
Assuming we started with 15 DNA molecules, how many DNA molecules are produced after 25 PCR cycles?
144
# **Recombinant DNA technology (Maths)**
How many PCR cycles produced 7230124899 DNA molecules?
145
# **Recombinant DNA technology (AO1)**
Doubling of DNA molecules each cycle;
but very low numbers to start with, so slower rate of increase but then exponential (faster rate) increase;
146
# **Recombinant DNA technology (AO1)**
Why does the number of DNA molecules produced eventually plateau after many PCR cycles?
DNA nucleotides being used up;
so less / nothing to make complementary strands;
OR
Primers used up;
so thermostable DNA POLYMERASE cannot start complementary strands;
147
# **Recombinant DNA technology (Maths)**
A DNA molecule consisting of a 8 base pair sequence was amplified 40 times using PCR.
Calculate the total number of base pairs in all the DNA molecules after 40 cycles of PCR.
148
# **Recombinant DNA technology (AO1)**
Benefits of recombinant DNA technology
**Replace faulty or lack of protein**
*(e.g. insulin in type 1 diabetes)*
**Gene therapy**
*(e.g. correct for mutation or lack of gene expression in an organsim)*
**Improve agriculture**
*(e.g. larger yields, disease resistance, drought tolerance)*
**Improve industrial processes**
*(e.g. produce larger quantities of enzymes more quickly OR improve enzyme-controlled rate of reaction)*
**Improve understanding of biological processes**
*(e.g. the regulation of gene expression, DNA replication, mitosis and cell death)*
149
# **Recombinant DNA technology (AO1)**
Concerns regarding the widespread use of recombinant DNA technology
**1.** Inserting new genes into a crop plant could disrupt other gene/s function creating toxic products within genetically modified food sources.
**2.** Introducing herbicide resistance genes to crop plants could result in transfer to wild species when they interbreed.
**3.** Technology may become concentrated in the hands of large corporations.
*(e.g. technology improves health outcomes but is not accessible to socially and economically disadvantaged groups).*
150
# **Recombinant DNA technology (AO1)**
What is gene therapy?
Introduction of healthy gene (which codes for functioning protein) into defective cells (caused by a mutation).
151
# **Recombinant DNA technology (AO1)**
Vectors for gene therapy in humans
Viruses
Liposomes
| Plasmids cannot be used to transfer genes into human cells
152
# **Recombinant DNA technology (AO1)**
Describe how a virus acts as a vector in gene therapy.
**1.** Insert isolated DNA fragment or gene of interest into viral genome.
**2.** Virus will insert this gene at the same time as its own genetic material into hosts cells during infection.
**3.** Host cell transcribes and translates gene that codes for functioning protein during replication
153
# **Recombinant DNA technology (AO1)**
Disadvantage of using viral vectors
**1.** May cause an immune response
e.g. formation of cytotoxic T cells / B cells / memory cells
**2.** Not all host cells are successfully infected with the genetically modified virus
154
# **Recombinant DNA technology (AO1)**
Advantage of using viral vectors
**1.** Can enter cells / infect cells / inject DNA into cells;
**2.** Targets specific cells
*(attachment proteins bind to receptors);*
**3.** Replicates in cells;
155
# **Recombinant DNA technology (AO1)**
Describe how a liposome acts as a vector in gene therapy.
**1.** Lipid soluble
**2.** So cross the phospholipid bilayer
(and release DNA / gene into cells)
156
# **Recombinant DNA technology (AO2)**
Suggest why the plasmids which contain a gene of interest from a spider - that codes for protein that produce stronger silk fibres - were injected into the eggs of silkworms, rather than into the cells of adult silkworms (2 marks).
**1.** Gene gets into all / most of cells of silkworm;
**2.** So gets into cells that make silk.
157
# **Recombinant DNA technology (AO2)**
The scientists ensured the spider gene - that codes for stronger silk fibre proteins - was expressed only in cells within the silk glands of the silkworm.
What would the scientists have inserted into the plasmid along with the spider gene to ensure that the spider gene was only expressed in the silk glands of the silkworms?
Promoter
158
# **Recombinant DNA technology (AO2)**
The scientists ensured the spider gene - that codes for stronger silk fibre protiens - was expressed only in cells within the silk glands of the silkworm.
Suggest two reasons why it was important that the spider gene was expressed only in the silk glands of the silkworms.
**1.** So that silk fibre protein can be harvested;
**2.** Silk fibre proteins in other cells might cause harm;
159
# **Recombinant DNA technology (AO2)**
Using the below information, explain why individuals who have been treated by gene therapy do not pass on the ADA gene to their children.
reproductive cells / gamete cells do not contain ADA allele / gene;
160
# **Genetic Fingerprinting (AO1)**
DNA [1] and DNA hybridisation are used to locate specific [2] of genes.
[1] probes
[2] alleles
161
# **Genetic Fingerprinting (AO1)**
What is a DNA probe?
- Short, **single strand** of DNA bases
- Bases **complementary** to specific DNA / allele / gene
- Attached to a fluorescent or radioactive label.
162
# **Genetic Fingerprinting (AO1)**
What is DNA hybridisation?
1. The single stranded DNA probe;
2. Binds to single stranded complementary DNA bases of DNA / allele / gene
| The double stranded DNA is a hybrid of probe and target DNA
163
# **Genetic Fingerprinting (AO1)**
What techniques can be used to detect the DNA probe?
Autoradiography / X-ray film for radioactive markers
UV light for fluorescent markers
164
# **Genetic Fingerprinting (AO1)**
Describe how a person could be screened for known harmful mutations of tumor suppressor genes BRAC1 and BRAC2 that increase the risk of cancer
(*5 marks*).
**1.** Extract DNA from cells in a sample
*(e.g. saliva, blood, skin)*
**2.** Use of PCR to amplify DNA;
**3.** Cut DNA using restriction endonucleases;
**4.** Separate DNA fragments (by size and charge) using gel electrophoriesis;
**5** Treat DNA so single stranded
*(e.g. via heat to break hydrogen bonds)*
**6.** Add DNA probes which bind via complementary base pairing to DNA base sequences for the mutations
**7.** Mutations identified by fluorescence / radioactivity
OR
Mutations identificatied using X-ray, autoradiography or UV light.
165
# **Genetic Fingerprinting (AO2)**
**1.** Carriers are heterozygous
**2.** Both have DNA that binds (about) half / 50% amount of probe (that non-carrier does);
**3.** Probe binds to dominant / healthy allele so only one copy of exon in their DNA.
166
# **Genetic Fingerprinting (AO2)**
The DNA probe the geneticist used was for an exon in the DNA, not an intron. Explain why (*2 marks*).
**1.** Introns do not code for amino acids / not in mRNA / not translated;
**2.** Mutations of these exons affect amino acid sequences that produce faulty protein / change tertiary structure of protein;
**3** So important to know if parents’ exons affected, rather than introns;
167
# **Genetic Fingerprinting (AO1)**
What does VNTR stand for?
**v**ariable **n**umber **t**andem **r**epeat
168
# **Genetic Fingerprinting (AO1)**
The probability of two individuals having the same VNTRs is very _______.
low
169
# **Genetic Fingerprinting (AO1)**
Location of VNTRs
In between genes
OR
Intergenic
170
# **Genetic Fingerprinting (AO1)**
**TRUE or FALSE:**
DNA probes can bind to repeat sequences within the VNTR
TRUE
171
# **Genetic Fingerprinting (AO1)**
Applications of genetic fingerprinting
**1.** Forensics
*e.g. matching DNA found at crime scene and suspect*
**2.** Paternity testing
*e.g. looking for overlapping VNTRs between parents and offspring*
**3.** Medical diagnosis
*e.g. detect a specific allele linked to a disease*
**4.** Genetic counselling during pregnancy
**5.** Plant breeding
**6.** Reducing inbreeding among individuals of an enderdangered species
172
# **Genetic Fingerprinting (AO2)**
**1.** More probe binding to gene A means more light;
**2.** DNA (with A) doubles each (PCR) cycle;
**3.** So light doubles / curve steepens more and more (each cycle) / curve goes up exponentially / increases even faster;
173
# **Genetic Fingerprinting (AO2)**
Use the below information to explain which person, H or G, was heterozygous, Aa?
(G because)
**1.** Heterozygous only has half the amount of probe for A attaching / only half the amount of DNA / allele A (to bind to);
**2.** So only produced about half the light / intensity (of H) (per cycle of PCR);
174
# **Genetic Fingerprinting (AO1)**
Explain how gel electrophoresis separates fragments of DNA
**1.** Move towards anode / positive end because negatively charged;
**2.** Different rates of movement related to size of fragment (i.e. how many base pairs it contains);
*e.g. shorter fragments move faster through the gel and towards the anode*
175
# **Genetic Fingerprinting (AO2)**
Lane 1 has DNA fragments of **known sizes/lengths**;
**Allows comparisons** (of position of viral fragment/s);
176
# **Genetic Fingerprinting (AO1)**
After gel electrophoresis, the DNA is treated to form single strands.
Explain why.
So DNA probe binds
(to complementary seequence)
177
# **Genetic Fingerprinting (AO1)**
**1.** Restriction endonucleases;
**2.** (Cuts DNA at specific) base sequence
OR Hydrolyse phosphodiester bonds
OR Hydrolyse at restriction site;
178
# **Genetic Fingerprinting (AO2)**
**1.** Each primer has a specific base sequence;
**2.** That is complementary (to allele r or R).
179
# **Genetic Fingerprinting (AO1)**
Explain why radioactive DNA probes are used to locate specific DNA fragments.
1. DNA invisible on gel / membrane;
2. Allows detection;
180
# **Genetic Fingerprinting (AO1)**
Describe genetic fingerprinting
(*5 marks*).
**1.** Extract DNA from cells in a sample
*(e.g. saliva, blood, skin)*
**2.** Use of PCR to amplify DNA;
**3.** Cut DNA using restriction endonucleases;
(leaving gene, allele, VNTR in tact)
**4.** Separate DNA fragments (by size and charge) using gel electrophoriesis;
**5** Treat DNA so single stranded
*(e.g. via heat to break hydrogen bonds) *
**6.** Add DNA probes which bind via complementary base pairing to DNA base sequences (for gene / allele / VNTR)
**7.** Gene / allele / VNTR identified by fluorescence / radioactivity
OR
Mutations identificatied using X-ray, autoradiography or UV light.
181
# **Genetic Fingerprinting (AO1)**
During genetic fingerprinting, the different short tandem repeat sequences (a type of VNTR) are separated by gel electrophoresis.
Give two features of these sequences which enable them to be separated by gel electrophoresis.
**1.** Number of nucleotides/repeats/bases/base pairs
OR Length/mass;
**2.** Negative charge;
182
# **Genetic Fingerprinting (AO1)**
Genetic fingerprinting using 'short tandem repeats' (STRs) requires amplification of the STRs using the polymerase chain reaction (PCR).
The short base sequences either side of a specific STR are known.
Explain the importance of knowing these base sequences in PCR.
**1. ** For primers;
**2.** To produce a complementary base sequence
OR primers provide starting sequence for DNA/taq polymerase;
