6.1.3 - Manipulating Genomes

Question 1

What is DNA sequencing?

Answer 1

Finding a nucleotide sequence for a gene or the whole genome

Question 2

What are the 2 types of DNA sequencing?

Answer 2

• Sanger sequencing
• High-throughput sequencing

Question 3

What are the 3 steps of Sanger sequencing?

Answer 3

• Create copies of DNA fragments by extracting a DNA sample and heating it to separate the 2 DNA strands. Then cut the DNA strands into DNA fragments and create many copies
• Create complementary DNA fragments by placing the DNA fragment in a mixture where DNA polymerases use DNA primers to attach to the DNA fragments and then make complementary DNA fragments by using DNA nucleotides. However if DNA polymerase uses a terminating DNA nucleotide instead it will stop it from adding further nucleotides. As a result many complementary DNA fragments are produced all ending with a different terminating DNA nucleotide.
• Analyse complementary DNA fragments by separating the DNA fragments by length and work out the original samples DNA sequence

Question 4

What are the advantages of high-throughput sequencing?

Answer 4

• Automated
• Very rapid
• Cheaper

Question 5

What are the 3 main benefits of DNA sequencing?

Answer 5

• It enables genome wide comparisons between individuals and species which reveals how closely related different individuals or species are
• Allows us to predict the amino acid sequences of genes to reveal the tertiary structure of the polypeptide the genes code for
• Useful for synthetic biology which modifies existing DNA sequences and these modified DNA sequences produce specific proteins which can then be used as drugs

Question 6

What is bioinformatics?

Answer 6

Bioinformatics is a field of biology that involves the storage, retrieval, and analysis of data from biological studies. These studies may generate data on DNA, RNA and protein sequences, and on the relationship between genotype and phenotype. Once a genome is sequenced, bioinformatics allows scientists to make comparisons with the genomes of other organisms using the many databases available which can help to find the degree of similarity between organisms which then gives an indication of how closely related the organisms are

Question 7

What is gel electrophoresis?

Answer 7

A technique used to separate molecules of DNA, RNA or proteins

Question 8

How are molecules of DNA and RNA separated?

Answer 8

By mass where shorter fragments have a lower mass and longer fragments have a greater mass

Question 9

How are proteins separated?

Answer 9

• By mass determined by the size of their R groups or the number of amino acids present
• By charge determined by the R groups

Question 10

How do you prepare a gel electrophoresis?

Answer 10

• Cut a line of holes called wells into a piece of agar gel
• Submerse the agar gel into a buffer solution
• Load the molecules you want to separate into one of the wells
• Place a negative electrode at the end of the gel with the wells and a positive electrode at the opposite end
• Apply an electric current moving from negative to positive electrode
• Multiple samples can be compared by filling multiple wells in the agar
• The different bands are seen by adding a fluorescent dye to the gel which glows under UV light

Question 11

How do the molecules get separated in gel electrophoresis?

Answer 11

The lighter or more negatively charged a molecule is the faster it will move across the gel so in a given period of time these molecules will move further than those that are heavier or less negatively charged

Question 12

What is gel electrophoresis used for?

Answer 12

• To separate DNA fragments for genome sequencing
• DNA profiling

Question 13

What is genetic engineering?

Answer 13

The process of isolating a gene from one organism and placing it into another organism. These organisms can then translate the added gene because the genetic code is universal and we can use it to modify animals, plants and microorganisms.

Question 14

What are the uses of genetic engineering?

Answer 14

• Gene therapy
• Modify plants such as soybeans to give them insect resistance
• Modify pathogens for research into developing new medical treatments
• Pharming where an animal’s DNA is altered so that they produce human proteins for medicine or they develop human diseases so new pharmaceuticals can be tested on them

Question 15

What is gene therapy?

Answer 15

When a patient’s DNA is altered to treat or cure a disease

Question 16

What are the 2 types of gene therapy?

Answer 16

• Somatic gene therapy
• Germ-line gene therapy

Question 17

What happens in somatic gene therapy?

Answer 17

A new gene is introduced into body cells called somatic cells specifically targeting cells in the tissues that need the treatment. However as somatic cells eventually die this treatment has short lived effects.

Question 18

What happens in germ-line gene therapy?

Answer 18

The germ is introduced into germ cells (sperm and egg cells). This means all the cells in the offspring will be altered and therefore the treatment has long term effects that will be inherited.

Question 19

What are the ethical issues associated with genetic engineering?

Answer 19

• Should we modify animals to act as models for human diseases?
• Should we put human genes into animals?
• When a company genetically modifies an organism they will patent this modification meaning the company can charge a large amount of money for the modified organism. For example, they can charge more for genetically modified seeds which can prevent poorer farmers from successfully growing crops

Question 20

What are the 2 ways DNA fragments can be produced?

Answer 20

• Restriction endonuclease
• Reverse transcriptase

Question 21

How does a restriction endonuclease produce a DNA fragment?

Answer 21

It cuts the DNA molecule at a specific set of bases known as a recognition sequence

Question 22

How does reverse transcriptase produce a DNA fragment?

Answer 22

It adds DNA nucleotides to an mRNA molecule which forms a new strand called complementary DNA. An enzyme then destroys the mRNA strand and a second DNA strand is built by DNA polymerase which results in a complete DNA fragment

Question 23

What are the steps of genetic engineering?

Answer 23

• A DNA fragment containing the gene is prepared using restriction endonucleases that produce sticky ends
• The DNA fragment is inserted into a vector (often a plasmid) which is done by using the same restriction endonuclease on the plasmid producing sticky ends that are complementary to the DNA fragment. The DNA fragments nucleotides are then joined to the plasmid’s nucleotides by DNA ligase which forms a recombinant plasmid
• The recombinant plasmids are inserted into bacteria which is called transformation and requires the addition of calcium ions and heat shock or electroporation which makes it easier for the plasmids to pass through the bacteria cell surface membrane
• Identify the bacteria which do contain a recombinant plasmid by beginning genetic engineering with a plasmid that contains an antibiotic resistance marker gene so once placed in the antibiotic any bacteria with the recombinant plasmid die
• Multiply bacteria creating a lot of the DNA fragment

Question 24

What are some of the issues during in vivo cloning?

Answer 24

• Sometimes the plasmid just rejoins to itself
• Sometimes the DNA fragments join to each other
• During transformation its likely that some bacteria just won’t take up a plasmid at all

Question 25

How do they avoid the antibiotic resistance marker genes being transferred to other disease causing bacteria?

Answer 25

Scientists use different marker genes such as fluorescent marker genes which cause bacteria to glow and enzyme marker genes which produce an enzyme that causes an observable effect

Question 26

What does the polymerase chain reaction do?

Answer 26

It produces copies of DNA fragments in a continuous cycle

Question 27

What does the polymerase chain reaction require?

Answer 27

• DNA fragment
• DNA nucleotides
• DNA polymerase (Taq polymerase)
• Primers

Question 28

What are the 3 stages of the polymerase chain reaction?

Answer 28

• The DNA fragment is heated to 95°C which breaks the hydrogen bonds between bases causing the strands to separate
• The temperature is reduced to 55°C to enable primers to join the complementary bases at the end of each strand
• The temperature is increased to 72°C and with the help of primers Taq polymerase adds complementary nucleotides to each strand until 2 new identical DNA fragments are formed

Question 29

What are short tandem repeats?

Answer 29

Variable numbers of repetitive non-coding DNA sequences that are directly next to each other. They can be found on many chromosomes within a single organism

Question 30

What does DNA profiling do?

Answer 30

It analyses and compares STRs between 2 or more individuals

Question 31

What are the steps of DNA profiling?

Answer 31

• Collect DNA from a tissue sample
• Add proteases to remove histones (DNA purification)
• Run PCR to amplify DNA
• Use restriction enzymes to produce DNA fragments containing STRs
• Run gel electrophoresis to separate STRs
• Add alkali to separate each fragment into single strands
• Add complementary DNA probes which then attach to the STRs making them easier to spot
• Compare STR patterns

Question 32

What are the uses of DNA profiling?

Answer 32

• Medical diagnosis to analyse the risk of inheriting certain diseases such as cystic fibrosis
• Forensic science to identify suspects of a crime
• Animal/plant breeding to identify closely related individual to prevent genetic disorders in the offspring
• Population genetics to determine genetic variation within a population