Gene Technology

Question 1

State the three methods for obtaining DNA fragments

Answer 1

Using restriction endonuclease
Using reverse transcriptase
Using a ‘gene machine’

Question 2

Describe how restriction endonuclease can obtain DNA fragments

Answer 2

Group of enzymes that n aurally occur in bacteria
They cut DNA at a specific sequence of bases (called a recognition sequence) by hydrolysing the phosphodiester bonds in the sugar phosphate backbone
They cut the DNA between opposite base pairs in a staggered structure. This creates sticky ends that can bind to other sticky ends with complimentary base pairing

Question 3

What are sticky ends?

Answer 3

Single stranded sections of DNA that form an overhang at the end of a double stranded molecule

Question 4

How is a gene machine used to obtain fragments of DNA?

Answer 4

Where genes are synthesised artificially:
- the amino acid sequence of a gene is determined
- the mRNA codons for each amino acids are searched
- the complimentary DNA triplets for the mRNA sequences are worked out and the gene is produced
-the genes are checked using standard sequencing techniques. Any with errors are rejected

Question 5

What are benefits of producing artificial genes with the gene machine?

Answer 5

Genes are free of introns and other non-functioning DNA- so they can be transcribed and translated

Question 6

After isolation of fragments of DNA, how is the number of DNA fragments increased?

Answer 6

Polymerase Chain reaction

Question 7

What are primers?

Answer 7

A short sequence of single stranded nucleotides, that have a specific base sequence

Question 8

What are the components of a thermal cycle used in PCR?

Answer 8

DNA fragments that need to be copied
Taq polymerase
Free nucleotides
Primers

Question 9

Explain the process of the polymerase chain reaction

Answer 9

In the thermal cycler, at 95*C the hydrogen bonds are broken and DNA double strand separates.
Temp is reduced to 55, primers attach to complimentary base sequences at the ends of the single strand
DNA polymerase attaches and the temp is raised to 72 (optimum temp for enzymes)
Free nucleotides attach to the single stranded templates via complimentary base pairing
DNA polymerase joins nucleotides together with phosphodiester bonds
DNA doubles in quantity

Question 10

How can reverse transcriptase be used to obtain DNA fragments?

Answer 10

Reverse transcriptase is an enzyme that converts single stranded mRAN to double stranded DNA.

It converts mRNA for the target gene into DNA. To create complimentary DNA fragments called cDNA

The cDNA can then be used to create recombinant DNA

Question 11

What are two advantages for using reverse transcriptase to produce a gene instead of removing DNA from an organisms genome?

Answer 11

mRNA has a higher concentration. Within cells so it is easier to isolate than genes

The cDNA that is produced will not contain non-coding introns, so the gene will be able to be expressed in prokaryotic cells

Question 12

What is gel electrophoresis?

Answer 12

A technique that separates different pieces of DNA based on their length

Question 13

Once gel eletrophoresis has separated pieces of DNA, what can they be used for?

Answer 13

Locating defective genes that are used against specific diseases, using gene probes

Analysing DNA fragments to produce a genetic fingerprint

Question 14

Describe the process of gel electrophoresis

Answer 14

DNA samples are amplified with PCR, and cut into fragments with restriction enzymes
Fragments placed into wells at one end of a thin bit of gel
An electric current is passed through the gel, the DNA fragments are attracted to the positive electrode
Molecules move through the gel, the shorter the lengths log DNA, then faster and further they move in a given time

Question 15

During gel electrophoresis, why are DNA fragments attracted to the positive electrodes?

Answer 15

DNA is negatively charged due to the phosphate ions

Question 16

What is used to mark the DNA fragments during gel electrophoresis?

Answer 16

Stationed with fluorescent chemicals
Radioactively labelled, and the. An x-ray film is used to identify their positions

Question 17

During gel electrophoresis, what are size markers and what are they used for?

Answer 17

A series of bands of DNA fragments of known sizes, that can be used to estimate the sizes of the unknown fragments by comparing the positions of their bands

Question 18

What is genetic screening and diagnosis?

Answer 18

Where a gene probe is used to identify if a gene or allele is present in a DNA fragment, after gel electrophoresis

Question 19

Explain what a gene probe is, and how it is used in genetic screening?

Answer 19

A gene probe is a single stranded short sequence of DNA nucleotides that have a complimentary base sequence to a specific gene or allele

The DNA from gel electrophoresis is made single stranded using an alkali.
The gene probe is added
The gene probe will bind to the target gene or allele if it is present in a DNA fragment, it will be labelled with a radioactive or fluorescent tag to it can be seen

Question 20

What genes/ alleles are most commonly screened for in genetic screening?

Answer 20

Oncogenes that may cause cancer
Sickle cell anaemia
Cystic fibrosis

Question 21

What is used to advise people on genetic screening?

Answer 21

Genetic counselling

Question 22

What is genetic fingerprinting?

Answer 22

A diagnostic tool that analyses the small differences in DNS bade sequences between individuals of the same species, by looking at non-coding DNA instead of coding DNA

Question 23

What DNA does genetic fingerprinting use and not use?

Answer 23

Does not use coding DNA
Does NOT use non-coding DNA found within genes (introns)
DOES use the non-coding DNA found between genes

Question 24

What are variable number tandem repeat?

Answer 24

Repetitive sequences of non coding DNA found between genes, used in genetic fingerprinting
Each person has a unique pattern

Question 25

Describe the technique used for genetic fingerprinting

Answer 25

Extraction- DNA is extracted from a nucleus, and amplified using PCR Digestion- restriction endonuclease cut recognition sites close to the variable number tandem repeats Separation- gel electrophoresis separates the DNA fragments, and double stranded DNA is turned into single stranded in alkali Binding to DNA probes- DNA probes, that are radioactive or fluorescent, bind to the variable number tandem repeats. Different repeats will cause a different amount of DNA probes to bind Visualisation- series of bands are seen by using the fluorescent and radioactive probes DNA from crime scenes can then be compared to suspects DNA to see if the patterns are identical

Question 26

What are the 4 uses of DNA fingerprinting?

Answer 26

Paternity tests- seeing if genetic relationships are seen Forensic science- crime scenes Medical diagnosis- sample of DNA can be compared with someone who has a disease to see if they are similar Plant and animal breeding- prevent inbreeding by seeing how closely some animals of the same species are related

Question 27

What is in vivo cloning?

Answer 27

The transfer of DNA fragments from one organism to another. The genetic code is universal, as well as transcription and translation being universal. Therefore the DNA can be translated within the organism

Question 28

Describe the process of in vivo cloning

Answer 28

The DNA fragment is prepared for insertion Insertion of the DNA fragment into a vector (which carries things between organisms) Transformation- the vector transfers then DNA into suitable host cells Identification of the transformed cells using gene markers    The transformed cells population grow and clone

Question 29

In vivo cloning- how is DNA prepared for insertion?

Answer 29

A promoter sequence is added to the start of the DAN fragment, to provide a binding sit for RNA polymerase A terminator sequence is added to the end, to release the RNA polymerase and end transcription

Question 30

In vivo cloning- what is used to transfer the DNA fragments from organism to organism, using vectors?

Answer 30

A vector transports the DNA into a host cell, using recombinant plasmids. The vectors contain recognition sits, so restriction enzymes can cut them open. They contain ampicillin resistant genes and green fluorescent protein genes- which are used as gene markers

Question 31

In vivo cloning- Describe the process of modifying plasmids to make recombinant plasmids

Answer 31

A restriction enzymes is used to cute the gene from the DNA fragments, this creates sticky ends The same restriction enzyme cuts the plasmids to create complimentary sticky ends Gene, plasmid and DNA ligase enzyme mixed in a test tube, gene and plasmid are joined by DNA ligand. Sticky ends are joined by phosphodiester bonds This is a recombinant plasmid, ready for in vivo cloning Sometimes the plasmid sticks ends rejoin without the gene

Question 32

In vivo cloning- why will only a small number of bacteria cells be ‘transformed’?

Answer 32

Some bacteria cells will take in the original plasmids Some bacteria will not take in any plasmids at all

Question 33

In vivo cloning- what are marker genes used for and how do they work?

Answer 33

They differentiate between the bacterial cells that take in in no plasmids, the original plasmids and the recombinated plasmids/ The green fluorescent protein (GFP) will only be expressed if a gene is inserted into the plasmid (in a recombinant plasmid). And so it will glow when it is exposed to UV light

Question 34

What is gene therapy?

Answer 34

A treatment for genetic diseases. Where the genotype is altered by supplementing specific alleles

Question 35

Describe the process of gene therapy

Answer 35

The non-defective allele is introduced into cells, so it is present with the defective allele: It is first inserted into a vector, which delivers the allele into the nucleus of the cell The cell transcribes the gene to mRNA mRNA is translated into a functional protein, genetic disease is cured

Question 36

What type of alleles does gene therapy work for, and why?

Answer 36

Recessive alleles only, because the defective alleles are still present, so if the original allele was dominant it would still be expressed even if a normal recessive allele is added.

Question 37

Give examples of how genes can be modified in agriculture

Answer 37

Genes that are resistant to pests, diseases and herbicides can be added to plant species Genes that develop a tolerance to extreme environmental conditions can be added to species

Question 38

What are direct uses of genetically modified bacteria?

Answer 38

Used to increase the quantity of antibiotics and the rate in which they are made To produce hormones such as insulin, oestrogen and testosterone

Question 39

What are some arguments for recombinant DNA technology?

Answer 39

Microbes and other cells can be modified to produce useful substances such as insulin GM crops can help to prevent diseases Gene therapy can be used to treat genetic diseases such as cystic fibrosis

Question 40

What are some arguments against recombinant DNA technology?

Answer 40

Cannot predict the risks when releasing GM organisms into the environment Useful genes may mutate into harmful genes Ethical issues- how far should we take gene technology?

Question 41

What is the human genome project?

Answer 41

A world wide project that determines the sequence of bases of a human genome.

Question 42

What are applications of the human genome project?

Answer 42

Screening fro mutations Pre-implantation screening Screening from genetic disorders such as huntingtons disease

Question 43

What are ethical arguments against the human genome project?

Answer 43

People can be discriminated against Misuse and ownership of the genetic information

Question 44

What is the role of reverse transcriptase in DNA fragmentation (1 mark answer)

Answer 44

Produces cDNA using mRNA

Question 45

What is the role of taq polymerase in PCR?

Answer 45

To create complimentary strands of DNA using DNA nucleotides