Gene Technology

Question 1

What has automated sequencing led to?

Answer 1

automated sequencing of genomes has led to discovery of many new species.
Some species may look very similar but analysis of their genome shows differences in the DNA bases and therefore differences in the mRNA bases and amino acid sequences.

Question 2

Genome to Proteome

Answer 2

If researchers know which genes are present, they can determine which proteins can be produced via gene expression.
It is therefore possible to determine the full range of proteins produced by cells (PROTEOME)

Question 3

Applications of genome sequencing

Answer 3

identification of potential antigens for use in vaccine production.

Question 4

What has knowledge of the genome led to?

Answer 4

increased study of non-coding DNA and regulatory DNA (e.g. transcription factors, siRNAs, miRNAs, tRNAs)
These make up most of the DNA.
For example, in humans, it is now estimated that 80% of this non-coding DNA is involved in regulating the expression of protein coding genes which only make 1% of our entire genome.

Question 5

What is recombinant DNA?

Answer 5

a cell having two or more sources of DNA.

This can be achieved by ISOLATING fragments of DNA and then INSERTING this DNA into another organism

(recombinant is also referred to as transgenic or a genetically modified organism (GMO))

Question 6

Why is recombinant DNA technology possible?

Answer 6

the genetic code is:
universal
non overlapping
degenerate

transcription and translation are also universal

Question 7

What are the 5 steps in recombinant DNA technology?

Answer 7

1. Isolation of genes
2. Insertion
3. Transformation (transfer into microorganisms)
4. Identification
5. Growth / cloning

Question 8

Isolation using reverse transcriptase

Answer 8

makes cDNA from mRNA

Free DNA nucleotides bind to single stranded mRNA template via complementary base pairing.
Reverse transcriptase joins DNA nucleotides together to form a single stranded cDNA molecule.
DNA polymerase is required to make cDNA double stranded

Question 9

Advantages of using reverse transcriptase

Answer 9

• mRNA is much easier to obtain
• introns have been removed (bacteria can’t splice)
• produce protein in large amount

Question 10

Isolation using restriction endonuclease

Answer 10

Different restriction endonucleases hydrolyse DNA at different specific recognition sequences because the shape of the sequence is complementary to the enzymes active site.
These recognition sequences are often palindromic whereby the base pair read is the same in opposite directions (DNA is antiparallel).

The DNA sample is incubated with the specific restriction endonuclease(s), which hydrolyses the DNA into fragments wherever the recognition sequence appears.

If the target gene has recognition sequences before and after the target gene, the fragments will contain the desired gene

Question 11

What are Restriction endonuclease ?

Answer 11

enzymes that hydrolyse DNA at specific recognition (base) sequences.

These are usually either side of a desired gene

Question 12

What happens if the recognition sequence for the selected restriction endonuclease occurs within the DNA fragment you want to isolate?

Answer 12

will cut the gene and it will not code for a functional protein

Question 13

Blunt and sticky ends

Answer 13

blunt= restriction enzyme cuts in the same positions in both sides

sticky= restriction enzymes does not cut in the same position

Question 14

Suggest why the restriction enzyme has cut the human DNA in many places but has cut the plasmid DNA only once.

Answer 14

• enzymes only cut DNA at specific base sequence
• occurs once in plasmid and many times in human DNA;

Question 15

How can a gene be expressed?

Answer 15

once a DNA fragment is isolated, it is possible to add a ‘promoter region’ which then allows the gene to be expressed once inserted into the bacterial plasmid.

Similarly, ‘a terminator region’ is also added to the fragment which stops transcription.

Question 16

Explain how modified plasmids are made by genetic engineering and how the use of markers enable bacteria containing these plasmids to be detected.

Answer 16

• isolate TARGET gene from organism
• using restriction endonuclease to get DNA
• produce sticky ends;
• use DNA ligase to join TARGET gene to plasmid;
• also include marker gene;
• add plasmid to bacteria to grow
• bacteria colonies not killed have antibiotic resistance gene and the TARGET gene;

Question 17

Isolation using the gene machine

Answer 17

1. Desired nucleotide sequence fed into a computer
2. Synthesis of oligonucleotides (short sequences of nucleotides)
3. Oligonucleotides are overlapped then joined together and made double stranded using the polymerase chain reaction (PCR)
4. Gene is inserted into a bacterial plasmid

Question 18

Advantages of the gene machine

Answer 18

• easily transcribed and translated by prokaryotes, as they have no introns in DNA
• faster (all enzyme catalysed reactions)
• more accurate

Question 19

Insertion of genes into a vector

Answer 19

• Isolated Target DNA fragment inserted into vector DNA by cutting open the vector DNA using the SAME restriction endonuclease that was used to isolate DNA fragment.
• Produces complementary ‘sticky ends’ between the ends of DNA fragment and cut ends of vector DNA.
• Target DNA fragment anneals to vector DNA by complementary base pairing between their ‘sticky ends’.
• DNA ligase used to join the DNA fragment and vector DNA at the sugar phosphate backbone. (ligation) forms phosphodiester bonds.
• forms recombinant DNA.

Question 20

Vector definition

Answer 20

A vector is a DNA carrier (i.e., bacterial plasmid or virus) used to transfer foreign DNA into cells.

Question 21

Transformation producing recombinant organisms

Answer 21

process by which recombinant DNA VECTOR is transferred into a host cell (Bacteria)
Host cells which take up recombinant DNA are referred to as recombinant organisms or transformed organisms.

Question 22

Plasmid vectors

Answer 22

circular pieces of DNA found in bacterial cells.

If bacterial cells are placed in solution with recombinant plasmids, they can be encouraged to take up plasmids from the solution under certain conditions

Question 23

Describe a plasmid

Answer 23

• circular DNA;
• separate from main bacterial DNA;
• contains only a few genes

Question 24

Outline a method for in vivo gene cloning

Answer 24

1. Cut desired gene from desired organism;
2. Using restriction endonuclease

1 using DNA polymerase;
2 cut plasmid open;
3 with same restriction endonuclease;
4 ref. sticky ends
5 use DNA ligase to join;
6 return plasmid to bacterial cells

Question 25

Identifying transformed host cells

Answer 25

Transformed cells / organisms must be identified because: 1: some vectors take up target DNA to become recombinant 2: some host cells become transformed, by taking up recombinant vectors. Only transformed host cells, which contain the ‘target gene’, will synthesise desired protein

Question 26

Marker gene definition

Answer 26

allows easy identification of cells that have taken up a genetically transformed plasmid

Question 27

Identifying transformed bacteria using antibiotic resistance genes

Answer 27

1. Some cells will not have taken up any plasmid at all. These cells are killed by both types of antibiotic. 2. Some cells will have taken up an “original” plasmid. These are resistant to both types of antibiotics. 3. Some cells will have taken up a “transformed” plasmid. These cells are resistant to one type of antibiotic, but not the second (because the 2nd gene has been cut and disrupted by inserting foreign DNA). The colonies of bacteria that survive the 1st antibiotic and are destroyed by the 2nd antibiotic are the cells you want.

Question 28

Cloning (in vivo via bacteria)

Answer 28

Once you have identified the transformed bacterial cells then you can culture these bacteria as they reproduce by binary fission to produce genetically identical cells with the desired ‘inserted’ gene. These organisms will produce large quantities of the desired protein

Question 29

Cloning (in vitro via PCR)

Answer 29

Polymerase chain reaction (PCR) is used to AMPLIFY (make millions of copies of a single fragment of DNA). The number of DNA molecules doubles with every cycle, making it rapid & efficient.

Question 30

Describe the process of PCR

Answer 30

1. Heat DNA to 95C which breaks (weak) hydrogen bonds 2. Add primers and Add nucleotides; 3. Cool to 50C to allow binding of nucleotides 4. Add thermo stable DNA polymerase; 5. Heat to 75C 6. DNA polymerase joins nucleotides together; 7. Repeat cycle many times;

Question 31

Primers

Answer 31

are short pieces of single stranded DNA with complementary base sequences to bases at start of DNA fragment you want to isolate. Primers also prevent DNA strands sticking back together and allows DNA polymerase to attach and join nucleotides together Join to 5' end

Question 32

Why is the DNA heat to 95°C during PCR?

Answer 32

* Produce single stranded DNA * Breaks WEAK hydrogen bonds between strands

Question 33

Why do you add primers during PCR?

Answer 33

* Attaches to start of the gene * Replication of base sequence from here; * Prevents strands annealing

Question 34

Calculating number of DNA strands after x number of cycles

Answer 34

2^n

Question 35

Calculating number of cycles from number of DNA strands

Answer 35

Log2(number of DNA molecules)

Question 36

Comparison between ‘in vitro’ and ‘in vivo’ DNA fragment cloning

Answer 36

Vivo- Can be used to produce protein or mRNA from the inserted DNA as well as the target DNA. Vitro- Can only be used to copy DNA. Vivo- slower Vitro- quicker Vivo- Modified DNA Vitro- DNA not modified

Question 37

PCR cycle graph

Answer 37

start= slow, DNA doubling each cycle middle= exponential phase, going up in high numbers end= plateau, limiting factor - conc. primers = less new DNA strands - conc. nucleotides = fewer nucleotides

Question 38

Suggest one reason why DNA replication stops in the polymerase chain reaction

Answer 38

* Limited number of primers/nucleotides; * DNA polymerase denatures

Question 39

Explain why ‘base-pairs’ is a suitable unit for measuring the length of a piece of DNA.

Answer 39

* DNA = 2 chains * Bases are a constant distance apart * each base-pair is same length

Question 40

Benefits of recombinant DNA technology

Answer 40

Develop medical applications: -produce large quantities of insulin Develop agricultural applications. - Produce crops that are resistant to disease and/or extreme weather

Question 41

Concerns regarding recombinant DNA technology

Answer 41

* Antibiotic resistance may spread owing to its use as a selection marker * Inserting new genes into a crop plant could disrupt other gene/s function creating toxic products within genetically modified food sources

Question 42

Suggest how a scientist would arrive at their hypothesis

Answer 42

looked for info on crop plants that grow at ↑ temp, that contain GB assumed making plants produce GB makes them resistant to ↑ temp

Question 43

Gene therapy using viruses as vectors

Answer 43

Viral DNA is cut using the same restriction endonuclease and joined to foreign DNA using DNA ligase. The virus then acts as a vector (DNA carrier). Viruses can cause an immune response and the formation of cytotoxic T cells and memory B cells. vector viruses are further modified to evade the immune response.

Question 44

Using liposomes as vectors

Answer 44

Liposomes are lipid droplets which can cross the phospholipid bilayer and releases target DNA into the cell. However, the DNA does not move into the nucleus and so does integrate into the genome of stem cells lining the airways, so new daughter cells will not have the functional gene. From this DNA, small amounts of mRNA can be produced

Question 45

Somatic gene therapy

Answer 45

DNA transfer to our normal body tissue

Question 46

Somatic limitations

Answer 46

* Not all cells take up new DNA * Not all cells express DNA allele * Only some tissue types are accessible i.e., lungs, needs to be repeated – as cells die replaced by cells with faulty DNA, * Multiple treatments may be needed * Body can produce an immune response to the vector

Question 47

Germ line gene therapy

Answer 47

DNA transfer to cells that produce eggs or sperm

Question 48

Germ line limitations

Answer 48

* effects of gene transfer are unpredictable and, even if the target disease was cured, further defects could be introduced into the embryo. * Denial of human rights. Individuals resulting from germ line gene therapy would have no say in whether their genetic material should have been modified. * Potential abuse. Germ line gene therapy could be used not only to eliminate disease, but also to enhance favourable characteristics and suppress unfavourable ones

Question 49

Genetic screening

Answer 49

1. sequence of nucleotides on mutated gene determined by DNA sequencing 2. fragment of DNA with complementary bases, to mutant allele of gene, produced 3. DNA probe formed by fluorescently labelling DNA fragment 4. PCR used to produce multiple copies of DNA probe 5. Probe added to single stranded DNA fragments from person being screened 6. if complementary fragments present, DNA probe taken up and dye will fluoresce. if complementary fragment not present, DNA probe will not fluoresce -When the DNA PROBE binds to its complementary target DNA, this is called DNA HYDRIDIZATION

Question 50

What does the presence of fluorescence conclude?

Answer 50

* Carrier of a recessive allele known to cause a genetic disorder * Contains mutations in known genes linked to increased risk of disease * Contains genes that code for proteins that predict positive or negative response to a drug dosage

Question 51

Gene probe definition

Answer 51

A short, single stranded DNA molecule with a complementary base sequence to DNA fragment

Question 52

What are the regions between genes called?

Answer 52

VNTR’s –VARIABLE NUMBER TANDEM REPEATS

Question 53

VNTR

Answer 53

Each individual has two copies of each VNTR one on each homologous chromosome. One copy inherited from each parent. Differences in the length of VNTRs can be analysed via GEL ELECTROPHORESIS

Question 54

Describe how genetic fingerprinting is carried out

Answer 54

1. DNA extracted from sample; 2. DNA hydrolysed into segments using restriction endonuclease; 3. Must leave VNTR’s intact; 4. DNA fragments separated using electrophoresis; 5. mixture put into wells on gel and electric current passed through 6. Immerse gel in alkaline solution so two strands of DNA separated; 7. Probe added 8. areas with probe identified using autoradiography

Question 55

Name one mutagenic agent.

Answer 55

* high energy radiation * benzene * x rays * uv (light);

Question 56

A deletion mutation occurs in gene 1. Describe how a deletion mutation alters the structure of a gene.

Answer 56

* removal of one or more bases * frameshift

Question 57

Describe how the bacteria containing the insulin gene are used to obtain sufficient insulin for commercial use

Answer 57

* use of fermenters; * provides nutrients plus suitable conditions for optimum growth * environmental factor; * reproduction of bacteria; * insulin accumulates and is extracted;

Question 58

mRNA may be described as a polymer. Explain why.

Answer 58

* Made up of many similar molecules

Question 59

Name three techniques used by scientists to compare DNA sequences.

Answer 59

* Polymerase Chain Reaction * DNA fingerprinting * Gel electrophoresis