Component 2 - Application of genetics Flashcards
What are the main aims of the Human genome project?
Determine the order of bases in the human genome
Identification of all the genes
Sequencing those genes and mapping their position on each chromosomes
Store the information on a database
Consider the ethical, social and legal issues which arise from obtaining information about the human genome
What are the 100K genome project aims?
Launched in 2012
Use new generation sequencing (NGS) to sequence 100,000 genomes from patients with cancer/rare diseases
What are the applications of the 100K genome project?
Scan a patients DNA sample for mutations an compare the gene sequences to the normal version of the gene
Carrier screening
Pre-natal testing-amnicentesis
What are some ethical concerns with the human genome project?
Tests for late onset diseases in presently healthy individuals/children
Risk of discrimination
Lab errors - misidentification and contamination
Social stigmatization and anxiety
Human cloning
What are some applications of genetic engineering?
Genes into bacteria to make useful proteins - insulin
Genes into plants and animals so they acquire new characteristics - disease resistance
Genes into humans so they no longer suffer genetic diseases
What is a gene?
A sequence of bases on the DNA that codes for a polypeptide
What is meant by recombinant DNA?
When the DNA of an organism is formed from different organisms
What is meant by transgenic?
Organisms which contain DNA from another organism
Describe the sequence of events in 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 is the purpose of restriction endonuclease?
Cuts the DNA at short, specific palindromic sequences (4-8 bases long)
What is the purpose of DNA ligase?
Joins the sugar phosphate backbone of DNA sections together
In a condensation reaction
How do restriction enzymes cut and reseal DNA molecules?
Cut DNA at specific base sequence (restriction site)
Hydrolyses the sugar-phosphate backbone of DNA
Gives a staggered cut, leaves some bases exposed (sticky end)
What are sticky ends?
Short sequence of unpaired DNA bases
What is the purpose of reverse transcriptase?
Enzyme used to synthesis single stranded DNA from mRNA template
What are the different ways we can obtain the required gene?
Can be synthesised by an automated polynucleotide sequencer
Use a DNA probe to identify the gene on DNA fragments and use restriction enzymes to cut the gene out, producing sticky ends
Obtain mRNA copies of the gene from cells - convert this mRNA to a single stranded copy of DNA using enzyme reverse transcriptase - DNA polymerase then converts this to a double stranded piece of DNA (cDNA) for putting into a plasmid
What is a DNS probe?
A single stranded piece of DNA complementary to part of the desired gene
What are some of the problems with cutting the gene from the DNA?
Difficult to late the required gene
The recognition sequence for the restriction enzyme could be within the gene - which could cut in half
If the recognition sequence is too far from the start of the gene - the DNA will contain INTRONS which the bacteria that is transferring the gene into cannot recognise or cut out
What are some of the problems in producing cDNA?
Locating the gene
Restriction enzymes cutting the gene into non-functional fragments
Introns being present
The need for post-transcriptional processing
How do we insert the gene into a vector?
DNA must be introduced into the cell by a vector
A plasmid is a small piece of circular DNA found in bacteria
Treat cells containing the plasmids with chemicals to dissolve their cell walls
Use ultracentrifugation to separate the plasmids from the cell debris
How do we produce recombinant DNA?
Use same restriction endonuclease to cut required gene and to cut plasmid DNA
Result in staggered cuts and sticky ends that are complementary
Use DNA ligase to join the donor DNA and vector DNA together
This creates recombinant DNA
What are the problems with mixing together cut plasmids, isolated gene and DNA ligase?
DNA ligase can combine some plasmids with the isolated gene
OR
Seal the cut plasmid back up
How can bacteria take up plasmids?
Mix the plasmids with bacterial cells
Only (1%) take up the plasmids
Induced by heat shocking and the addition of calcium salts
These bacteria are now transformed
What is meany by transformation?
The taking up of DNA from outside the cell
What are some of the problems of bacteria taking up plasmids?
Produces 3 types of bacteria:
Bacteria which have not taken up any plasmids
Bacteria which have taken up plasmids without the donor DNA (resealed)
Bacteria which have taken up the recombinant plasmid
