11 - Application of Reproduction and Genetics (C2) Flashcards
What did the 100K Human Genome Project find out?
- The order of bases in the human genome
- The identification of some genes, their sequencing and mapping
What is a stem cell?
An undifferentiated cell capable of dividing to give rise to cells which can develop into different types of specialised cells
What are advantages of using stem cells?
- Speed of production
- Large scale production
- Production of genetically identical cells / organisms
What are disadvantages of using stem cells?
- In mammals the technique is very expensive and unreliable
- In plant disease, entry of pathogens may cause problems
- Inadvertent selection of disadvantageous alleles
- Long term / unforeseen effects such as premature ageing
What are the 2 methods of replacing defective genes?
- Somatic cell therapy
- Germ line therapy - hasn’t yet been performed
What does somatic cell therapy not prevent?
Defective genes from being passed on
What is DMD? (Duchenne Muscular Dystrophy)
- A recessive, sex-linked condition, affecting males
- Sufferers can’t produce functional dystrophin, an important structural component of muscle tissue
- Causes wasting of the muscles, so sufferers often use wheelchairs by their teenage years
- Low life expectancy (27)
What are sticky ends?
Unpaired bases at the ends of DNA
What problems does producing cDNA help overcome?
- Locating the gene
- Restriction enzymes cutting the gene into non-functional fragments
- The presence of introns
- The need for post-transcriptional processing to produce functional mRNA
What are 2 examples of GM crops?
- Tomatoes
- Soya
What are some benefits associated with the production of GM crops?
- Superior keeping qualities
- Higher yield
- Substantial reduction in pesticide use on crops engineered for resistance to fungal pathogens and insect attack
What are some concerns associated with the production of GM crops?
- Dispersal of pollen from crops engineered for herbicide resistance to wild relatives
- Unknown effects of eating new protein produced in the crop
- Reduction in biodiversity
What are STRs? (Short Tandem Repeats)
Blocks of repeated nucleotides in introns
What does PCR (polymerase chain reaction) do?
Amplifies small sections of DNA rapidly to create a large enough sample for extensive research
What does PCR allow?
Allows tests to be carried out on very small samples accurately and more rapidly regardless of the age of the sample
What is the process of PCR?
- DNA is heated to 95 degrees
- Sample is cooled to 50-60 degrees
- Heated to 70 degrees
- For each strand of DNA, 2 identical strands are produced
- The resulting PCR product can be run and visualised on electrophoresis gel
Why in PCR is the DNA heated to 95 degrees?
To separate the 2 strands
Why in PCR is the DNA sample cooled to 50-60 degrees?
To allow the primers to bind to the DNA strands (annealing)
Why in PCR is the DNA sample heated to 70 degrees?
To allow a thermally stable DNA polymerase (Taq) to add complementary nucleotides by forming the phosphodiester bonds in the sugar-phosphate backbone
What does gel electrophoresis do?
Separates DNA fragments according to size
What is gel electrophoresis gel made from?
Agarose, which contains pores in its matrix
What is the process of gel electrophoresis?
- DNA samples are loaded into wells at one end and a voltage is applied across the gel
- Add a mixture of DNA fragments of known lengths to build a DNA ladder
- DNA is attracted to the positive electrode due to its negative charge on the phosphate group
- Smaller fragments find it easier to migrate through the pores in the gel so they travel further than large fragments in the same time
How can fragment size of DNA be determined in gel electrophoresis?
By running a DNA ladder (which contains fragments of known size) alongside samples in the gel
What is recombinant DNA?
When the DNA within an organism includes one or more genes added by man via genetic engineering
What does DNA ligase do?
Joins (anneals) sticky ends
What is meant by ‘sequencing’ the human genome?
Determining the exact order of the base pairs in a segment of DNA
What is needed in PCR and why?
- Taq Polymerase - catalyses the synthesis of a complementary strand
- Buffer - DNA sample is dissolved in this
- Thermocycler - device which ensures rapid temperature change
- Primers - bind to the DNA strand, signalling taq polymerase to start replication
- Nucleotides - mixed with the DNA sample
Why would DNA polymerase from human sources not be suitable for use in a PCR machine?
As the enzyme would denature at these high temperatures
Why can’t species of plants be identifies from xylem material using PCR and DNA profiling?
DNA of plants is too different to that of humans so the PCR and DNA profiling methods don’t work
What are 2 methods of isolating DNA fragments for genetic engineering?
- Using restriction enzymes
- Using reverse transcriptase
What is the process of using restriction enzymes in genetic engineering?
- Gene is cut from genome using restriction enzymes isolated from bacteria, leaving sticky ends
- The endonuclease catalyses a hydrolysis reaction which breaks the sugar phosphate backbone of the DNA
- Once isolated the gene will be inserted into an appropriate plasmid that is cut using the same restriction enzymes used to produce complementary sticky ends
- Ligase forms phosphodiester bonds between sugar phosphate backbones
- The plasmid is now referred to as recombinant DNA, and will act as a vector to introduce the DNA into an appropriate bacteria cell
What is the process of genetic engineering?
- Obtain the required gene in a DNA fragment
- Insert this DNA fragment into a vector
- The vector carries the gene into a suitable host cell
- The recipient expresses the gene through protein synthesis
- Identification of the host cells that have taken up the gene by the use of gene markers
- Growth of the transformed host cells
What conditions must cells differentiating into specialised tissues have?
- Oxygen, nutrients, growth factors
- Correct pH, humidity and WP
Why is tissue engineering done?
To produce bio artificial tissues and organs that can be used to replace or repair damaged or defective tissue
What cells are used for atuologous tissue engineering?
Induced pluripotent stem cells
What cells are used for allogenic tissue engineering?
Embryonic stem cells
Why is using adult stem cells better?
There is no risk of the tissue being rejected by the immune system
What are some advantages of recombinant DNA technology?
- There’s no limit to the amount of protein that can be synthesised cheaply
- No need to extract proteins from mammalian organs
- Quick to change amount to demand
- Proteins made are identical to those made by humans so no risk of allergic reactions
What are some disadvantages of recombinant DNA technology?
- Complicated
- Difficult to identify genes from entire human genome
- Not all eukaryotic genes can be expressed in prokaryotic cells
- Using antibiotic resistance genes to identify transformed cells could accidentally introduce these genes into human pathogens
What are some ethical consequences of genetic testing?
- Testing for late onset diseases in presently healthy individuals causes anxiety when there is no cure
- Risk of social stigmatisation
- Risk of discrimination by insurers or employers
- Lab errors could cause misdiagnosis
What are weaknesses of PCR?
- DNA is at risk of contamination (in the sample or in the machine)
- Errors occur during polymerisation
- Polymerisation may be inhibited by contaminants (e.g. Hb breakdown products, dye from blue denim)
- After 20 cycles rate slows (as enzymes denature and the conc of primers falls below that of the separated strands, which tend to recombine)
What is the process of genetic fingerprinting?
- Multiply DNA from hypervariable regions via PCR
- Cut with restriction enzymes
- Separate the fragments by electrophoresis
- Transfer to a nylon mesh and add radioactive probes
- Place on photographic film for autoradiography
What are advantages of genetic fingerprinting?
- Can readily identify the guilty
- Can obtain a sample non-invasively
- Could set up a universal database to rapidly identify suspects
What are disadvantages of genetic fingerprinting?
- It’s possible to tamper with a sample to give false results
- A universal database is an invasion of privacy and could be used by employers or insurers to identify people with health problems
What is gene therapy?
- Where a faulty gene is modified or replaced so that a functional protein is produced in humans
- Helps relieve symptoms associated with a genetic disorder
What is somatic cell gene therapy?
Replacing faulty genes with correct copies in only certain tissues
Why is germ line gene therapy controversial?
- Involves the gene being inserted into an embryo or gamete. All the new cells formed would contain new gene and be passed on
- Modifying babies for non-medical reasons, playing God
- No consent from baby
What is DMD caused by?
Frame shift mutations within the dystrophin gene
What are totipotent cells?
Cells that can form all other types of cells
What are multipotent cells?
Cells that have the ability to self-renew by dividing
What does allogenic mean?
Cells come from a different individual of the same species
What are advantages and disadvantages of using embryonic stem cells?
+ Totipotent
- Allogenic - hence may be rejected by immune system
- Unethical
What are advantages of using induced pluripotent stem cells?
+ No moral problems
+ Limitless different genomes can be made
+ Autologous - hence no risk of rejection by immune system
What are main uses of stem cells?
- Tissue engineering
- Cell based therapy
- Screening for new drugs
- Study of development and differentiation in normal cells compared to those with genetic disorders
What happens in tissue engineering?
Stem cells colonise a porous scaffolding which later biodegrades leaving replacement tissues
- Scaffold provides nutrients, O2, growth factors to the cells and ensures correct shape of developed tissue
What are bacteria treated with in order to isolate their plasmids?
- EDTA to destabilise the cell walls
- Detergent to dissolve the phospholipid bilayer
- Sodium hydroxide to make an alkaline environment that denatures the membrane proteins
