Unit 8 Genome Projects

Question 1

Outline usefulness of genome projects in simpler organisms

Answer 1

• As scientists could identify proteins that derive from the geneticcode
• They could then identify potential antigens to use in the vaccine

In more complex organisms, the presence of non-coding DNA and
of regulatory genes means that knowledge of the genome cannot
easily be translated into the proteome.

Question 2

Outline usefulness of genome projects in complex organisms

Answer 2

• Presence of non-coding DNA andof regulatory genes means that knowledge of the genome cannot easily be translated into the proteome.

Question 3

Parts of module

Answer 3

Question 4

What does recombinant DNA technology involve

Answer 4

• Recombinant DNA technology involves the transfer of fragments of
  DNA from one organism, or species, to another.
• Transferring DNA fragments between/within species is possible because the genetic code is universal, and transcription and translation work the same in all organisms too.

Question 5

Outline 1 way fragments of DNA can be collected

Answer 5

METHOD creates DNA fragments from mRNA using the enzyme reverse transcriptase
- Enzyme naturally occurs in viruses, such as HIV, and it makes DNA copies from mRNA.
- Cell that naturally produces the protein of interest is selected.
- These cells should have large amounts of mRNA for the protein.
- Reverse transcriptase enzyme joins DNA nucleotides with complementary bases to the mRNA sequence.
- Single-stranded DNA is made (cDNA) and too make this DNA fragment double-stranded, the enzyme DNA polymerase is used.
cDNA is intron-free

Question 6

Outline a 2nd way fragments of DNA can be collected

Answer 6

RESTRICTION enzymes to cut fragments desired gene
- Restriction endonucleases are enzymes that cut up DNA andhey naturally occur in bacteria as a defence mechanism.
- Many restriction enzymes that have an active site complementary in shape to a range of different DNA base sequences, described as recognition sequences.
- Each enzyme cuts the DNA at a specific location.
- Some enzymes cut at the same location in the double-strand and create a blunt end, whilst other enzymes cut to create staggered ends and exposed DNA bases.
- The exposed staggered ends are palindromic and referred to as ‘sticky ends’ because they have the ability to join DNA with complementary base pairs.

Question 7

Outline a 3rd way fragments of DNA can be collected

Answer 7

CREATING Gene in Gene Machine
- Scientists first examine the protein of interest to identify the amino acid sequence, and from that work out what the mRNA and DNA sequence would be.
- The DNA sequence is entered into the computer, which checks for biosafety and biosecurity
- The computer can create small sections of overlapping single strands of nucleotides that make up the gene, called oligonucleotides.
- Oligonucleotides can then be joined to create the DNA for the entire gene.
- PCR is used to amplify the quantity of DNA and to make it double-strand & process makes intron-free DNA so that it can be transcribed in prokaryotic cells

Question 8

Outline the vivo technique for amplifying DNA

Answer 8

• Restriction endonuclease enzymes are used to cut out the gene (DNA fragment) of interest and cut at recognition sites leaving sticky ends.
• Promoter region must be added at the start of the DNA fragment and Terminator region added at the end
• Same Restriction endonuclease cuts plasmid leaving sticky ends which are complementary ends
• DNA ligase joins DNA and plasmid together.

Question 9

Outline the role of tranformation in vivo technique

Answer 9

• Transformation involves mixing the recombinant plasmids and bacterial cells in a solution of calcium ions
• Scientists must identify which bacteria successfully took up a recombinant plasmid

Question 10

Outline the vitro technique for amplifying DNA
(PCR)

Answer 10

• Requires DNA fragment, DNA polymerase, DNAnucleotides and
  primers
• Heat to 95 °C to break hydrogen bonds and separate DNA strands
• Reduce temperature TO 55 DEGREEES so primers bind to DNA strands;
• Increase temperature, DNA polymerase joins complementary free nucleotides an

Question 11

Define marker genes and the 3 types

Answer 11

• Marker genes on the plasmid can be used to identify which bacteria successfully took up the recombinant plasmid.
  3 different marker genes used are:
  1. Antibiotic resistance genes
  2. Genes coding for fluorescent proteins
  3. Genes coding for enzymes

Question 12

Describe how marker genes work

Answer 12

To identify if the plasmid was taken up, the bacteria are grown on agar plates containing antibiotics, such as ampicillin.
- If the bacteria took up the plasmid then they will have the gene for ampicillin resistance and will survive.
- DNA fragment is deliberately inserted in the middle of a marker gene; this could be a gene for resistance to another antibiotic or a fluorescent protein gene.
- Those that do fluoresce under UV light must contain a non-recombinant plasmid, but those that do not fluoresce must have the recombinant plasmid.

Question 13

Outline the use of labelled DNA probes to locate specific alleles of genes

Answer 13

• DNA probes are short, single-stranded pieces of DNA that are labelled radioactively or fluorescently so that they can be identified.
• This is used to locate specific alleles of genes and to screen patients for heritable conditions, drug responses or health risks.
  PROCESS
• A sample of patient DNA is removed and heated to make it single-stranded.
• Mixed with DNA probes that have been created to be complementary to a range of different alleles for genes
• If the patient has that allele, their DNA will bind with the probe, this can be identified using autoradiography

Question 14

Outline the use of DNA hybridisation to locate specifc allels on genes

Answer 14

• This is when DNA is heated to separate the double helix into single-strands, and this is then mixed with complementary sequences of single-stranded DNA (the DNA probe).
• Once cooled, the complementary strands will anneal
• This method is used in medical diagnosis to identify if someone has a copy of a particular allele within their DNA.

Question 15

Outline The use of this information in genetic counselling and personalised
medicine

Answer 15

• Screening for the presence of particular alleles allows doctors to select medicines and personalise health advice based on your genotype.
• Some drugs are more or less effective depending on the alleles you have, so this enables more effective and cost-effective treatment.
• Genetic counselling is a type of social work giving people advice and information following the screening of disease using alleles.

Question 16

Outline VNTRs

Answer 16

• 95% of human DNA is made up of introns which consist of many variable number tandem repeats (VNTRs).
• The probability of two individuals having the same VNTRs is very low, however, the more closely related you are the more similar the VNTRs are.

Question 17

Outline the first 2 stages of genetic fingerprinting

Answer 17

1. Extraction
   Extracting the individuals DNA
2. Digestion
   Cutting the DNA down into fragments
   Use Restriction Enzymes that cut just outside the VNTR (leaves the VNTR of the
   introns)

Question 18

Outline the last 3 stages of genetic fingerprinting

Answer 18

3. Separation
   Separate out the DNA fragments by gel electrophoresis
   Add alkali to make the separated fragments single stranded
   Transfer the fragments to a nylon membrane by Southern Blotting
   Add UV light so the DNA fragments set
4. Hybridisation
   Add radioactively labelled DNA Probes complementary to the DNA fragments
5. Development
   Add photographic film and take an x-ray to produce the banding pattern picture

Question 19

Outline the uses of genetic finger printing

Answer 19

• Forensic science to place suspects are crime scenes
• Medical diagnosis
• Ensure animals and plants not closely related before being bred