Manipulating Genomes Flashcards
What is a genome?
The entire genetic material that an organism contains
What are genes?
Sections of DNA on chromosomes
What are introns?
Non-coding sections of DNA
What are tandem repeats?
Repetitive sequences of DNA
Where are tandem repeats found?
Within introns
What is a variable number tandem repeat (VNTR)?
Short nucleotide sequences, organised as a tandem repeat
What are short tandem repeats?
Smaller repeated nucleotide sequences
Describe the method for DNA profiling
- Extract DNA
- Purify with protease to remove histones
- Cut into fragments with restriction enzymes
- Gel electrophoresis
- Nylon membrane placed on gel draws top strand of DNA onto nylon - southern blotting
- Strands fixed in place by UV/heat
- DNA probes added match intron base sequence = hybridisation
Why do restriction enzymes cut DNA at specific base sequences?
- Shape of enzyme active site complementary to shape of a particular sequence of bases in DNA
- ESC can form
- Relevant bonds can be broken (H bonds between bases and phosphodiester from backbone)
How does gel electrophoresis work?
- DNA fragments put into a well in a block of gel
- Along with loading dye (DNA visible)
- Placed in alkaline buffer solution (regulates pH)
- Electric current passed through gel
- DNA fragments move towards positive end (phosphate groups = - charge)
- The smaller the fragments, the further it moves
What is southern blotting?
Designed to locate a particular sequence of DNA within a complex mixture
What are DNA probes?
Single-stranded DNA used to detect presence of complementary nucleic acid sequences by hybridisation
What is hybridisation?
Single-stranded DNA/RNA molecules join to complementary DNA/RNA
Usually, how many different STR’s are looked at when doing a DNA profile?
At least 12
Why are at least 12 different STR’s looked at when doing a DNA profile?
- There is a slight chance that 2 people could have the same pattern for one particular STR in their DNA
- Chances of have the same pattern for 12 different STR’s is much lower
What is a polymerase chain reaction?
A method to amplify a DNA sample by making multiple copies of it for analysis
What “ingredients” are needed for PCR?
- Original tiny DNA sample
- Excess of free nucleotides
- Primers
- DNA Taq polymerase enzyme
What are primers?
Short pieces of single-stranded DNA which start the copying process
Describe the steps in PCR
1- Denaturing phase
- temp inside machine = 90°-95°C for 30 secs
- breaks H bonds between strands
2- Annealing phase
- temp decreased to 55°-60°C
- primers join to both ends of single DNA strands
3- Extension phase
- temp increased to 70°-75°C for 1 min
- optimum temp for DNA Taq polymerase to work
- adds bases to primers, extending complementary strands
What type of DNA polymerase is used in PCR?
Taq polymerase that isn’t denatured by high temps
Why might you not get as many copies of the DNA made as expected?
- May not be enough primers
- Primers may not attach to all DNA strands
- Insufficient nucleotides available
Give 3 uses of DNA profiling
1- Criminal investigations
2- Prove paternity of a child
3- Identify individuals at risk of disease
What is DNA sequencing?
The process of determining the nucleic acid sequence
What is the Human Genome Project?
An international scientific research project with the goal of determining the sequence of nucleotide base pairs that make up human DNA
Describe the principle of DNA sequencing (interrupted PCR)
- DNA is mixed with a primer, DNA polymerase, nucleotides and terminator bases
- Undergoes PCR where the DNA separates into single strands and primers anneal
- Nucleotides are added to the new DNA strand, some being terminator bases. Each time a terminator base is added, a strand terminates until all possible chains are produced
- DNA fragments are separated by electrophoresis
- Lasers detect colour and sequence
- Computer analysis of all data gives original DNA sequence
What are terminator bases?
- Modified versions of A/T/C/G
- Labelled with different coloured fluorescent markers
- Doubly de-oxidised (2 O2 atoms less)
What happens if a terminator bases attaches to the fragments of DNA being copied?
DNA strand can’t grow any longer
What is Next Generation Sequencing?
Generates and analyses millions of sequences per run, allowing researchers to sequence, resequence and compare data at a rate previously not possible
How has gene sequencing allowed for genome-wide comparisons between individuals and between species?
- Analysing DNA sequence is a more accurate way of classifying organisms
- The more similar the DNA base sequence, the more closely related
How had gene sequencing allowed for the sequence of amino acids in polypeptides to be predicted?
- Proteomics
- If scientists can work out primary sequence, they can predict (by looking at R groups) how it will arrange itself in secondary and tertiary structures, which then allow predictions about its final 3D shape
In what 2 ways is proteomics possible?
1- Spliceosomes
2- Protein modification
What is a promoter?
A region of DNA that initiates transcription of a particular gene
Give 3 reasons for genetic engineering
- Make resistant crops
- GM pathogens
- Pharming
Describe the steps in genetic engineering
- Obtain required gene (mRNA or probes)
- Restriction enzymes are used to cut open vector
- Plasmid cut with same restriction enzymes
- Sticky ends match up
- Plasmid and DNA mix with DNA ligase to join sugar phosphate backbone together = recombinant DNA
- rDNA inserted into bacteria via heat shock, electroporation or electrofusion
- Use marker genes to identify bacteria
Describe the 2 ways you can obtain the required gene in genetic engineering
Using mRNA
- Add reverse transcriptase
- Converts mRNA to a single strand of DNA (cDNA)
- DNA made double stranded by adding free nucleotides, primers and DNA polymerase
Using a DNA probe
- Use heat to make DNA single-stranded
- Add a probe, complementary to gene you are looking for
- Cut out gene using restriction enzymes
Describe the features of a restriction site
- 4-6 b.p long
- Usually palindromic (reads the same in 5’-3’ and 3’-5’
- Some of them leave blunt ends whilst others leave staggered ends
Describe how transformed bacteria are identified
Marker genes
- After GE, 3 types of bacteria are present = no plasmid, with plasmid and with rDNA
- Grow bacteria on ampicillin culture and bacteria with no plasmid die as they have no resistance
- Shine remaining 2 bacteria under UV light and those with rDNA don’t glow as their gene for bioluminescence
has been spliced
Describe heat shock
- Temp of culture lowered to 0 degrees
- Rapidly raided to 40 degrees
Describe electroporation
- Small current applied to bacteria makes membrane porous
Describe electrofusion
- Electric current applied to bacteria increases the rate of uptake
Give an example of GM plant
GM soy beans
- Contain gene that codes for protein that is toxic to insects
Describe the advantages of GM crops
- High yield
- Less pesticide spray
- Can be more nutritious
Describe the disadvantages of GM crops
- Encourages monoculture
- Can interbreed with wild plants, creating weed killer resistance
- Some people don’t eat GM crops
What is pharming?
Creating pharmaceuticals from animals
What are the advantages of pharming?
- Drugs more readily available
- Large-scale drug production
What are the disadvantages of pharming?
- Potential animal side effects
- Animals become assets with no feelings
What is the purpose of GM pathogens?
For disease treatment and research
What is the advantage of GM pathogens?
- Previously untreatable diseases are treatable
What are the disadvantages of GM pathogens?
- Scientists could become infected
- Could revert back to original form and cause outbreak
- Could lead to biological warfare
What are the advantages of placing legal copyrights on GM ideas?
- Scientists now hurry to become first to create GM ideas
- Owner generates income
What are the disadvantages of placing legal copyrights on GM ideas?
- Poor farmers may not be able to afford GM products
What is bioinformatics?
The development of software needed for organising and analysing biological data
What is computational biology?
Using software to create theoretical models of biological models
What are the 2 types of gene therapy?
Somatic therapy and Germ line therapy
What is somatic gene therapy
- Alters alleles of somatic DNA
- Disease passed to offspring
- As modification cannot be passed on to offspring
What is germ line gene therapy?
- Alters alleles in gametes
- GM is passed to offspring (currently illegal on humans)
What are the 3 types of gene therapy vector?
- Viruses
- Liposomes
- Artificial chromosomes
What are the advantages of gene therapy?
- Increases quality of life for those with genetic disease
- Prolongs life for those with genetic disease
- Germ line can prevent offspring getting disease
What are the disadvantages of gene therapy?
- Technology may be used for non-medical purposes
- Expensive
- Potential to cause more harm than good
