Chapter 21 - Genome projects and gene technologies Flashcards
(16 cards)
How do we locate a gene
using a gene probe which consists of a length of DNA complementary to the desired gene and a marker particle. In order for the gene probe to attach H-bonds need to be broken by heating
What are the 3 methods of obtaining a gene
using restriction endonuclease enzymes
using reverse transcriptase enzymes on mRNA
reverse engineering a polypeptide
what is the function of restriction endonucleases
to cut DNA at specific recognition sequences. These recognition sequences are 6 bases long and are palindromic. these enzymes are complementary to the recognition sequences
what are sticky ends
when a restriction endonuclease cuts a length of DNA there will be bases that are not paired with their complementary bases forming sticky ends from hydrogen bonds of these exposed bases
the more exposed bases the stickier (better) the sticky ends
what are blunt ends
when there are no exposed bases from the use of some restriction endonucleases (blunt end are not very useful)
drawbacks of using restriction endonucleases
- you need to know the base sequence of the desired gene
- you need to use a gene probe
- you need to find the right restriction endonuclease enzyme with the right recognition sequence and sufficient sticky ends
how to obtain a gene from using reverse transcriptase on mRNA
you need to know which cell produces the desired protein to use this method
- extract mRNA from cell’s cytoplasm and use reverse transcriptase enzyme to from complementary strand of DNA
- heat to break H-bonds and remove mRNA strand
- use DNA polymerase enzyme to form the other strand of DNA
- this forms complementary DNA or cDNA
- cDNA is useful as it doesn’t contain introns only exons
- (need to add a promoter and a terminator if you want the gene to be transcribed)
how to obtain a gene from reverse engineering a polypeptide
used when you have the protein that you want (occurs in a gene machine)
use the amino acid table to find a base sequence of DNA that would code for the desired polypeptide
this may not be the same as the gene that codes for that polypeptide as the genetic code is degenerate
* (need to add a promoter and a terminator if you want the gene to be transcribed)
What are the two methods of gene amplification
In vivo amplification - using bacteria
in vitro amplification - using polymerase chain reaction
Gene amplification by in-Vivo amplification
- obtain a plasmid by placing bacteria into pure water causing it to burst
- Obtain the desired gene using any method
- Obtain an antibiotic resistance gene - acts as a marker gene
- Use the same restriction endonuclease to place desired and antibiotic resistance gene into a plasmid so that the sticky ends match
- add promotor and terminator if not already present
- This forms a recombinant plasmid
- DNA ligase enzyme will reform the phosphodiester covalent bonds
- induce stress (salt,heating) so the recombinant plasmid is taken up by bacteria
- add antibiotic do induce a selection pressure so only bacteria with recombinant plasmid remain
- bacteria divides by binary fission
- either extract genes from plasmid or purify proteins produced
What is in the reaction mixture in PCR (polymerase chain reaction)
- desired gene - to by replicated
- excess DNA primers - to allow DNA polymerase to form the other complementary strand. Primers will be complementary to the 3’ end of DNA
- thermostable DNA polymerase - to from complementary strand
- excess free nucleotides
Gene amplification by in-Vitro amplification
- Set up reaction mixture
- heat to 94°C to beak H-bonds - denaturation
- cool to 52°C to allow DNA to associate with primers - annealing
- heat to 72°C to allow thermostable DNA polymerase to work
- repeat the process - each round of PCR double the amount of genes
method for gel electrophoresis
- extraction of DNA sample (PCR to amplify,obtaining a gene)
- digestion with restriction endonuclease enzyme into fragments
- load fragments onto a well cut into the top of a block of agarose gel
- run electricity into agarose block
- flow of electrons will push the fragments
- separation based on size and charge of DAN fragments
- hybridisation (binding) of gene probes to identify locations of specific sequences
- development using x-ray if necessary
- this forms a genetic fingerprint/profile
- compare to other genetic fingerprints
uses of genetic fingerprinting
forensics, genetic relationships, genetic diversity, plant and animal breeding, medical diagnosis
What is DNA profiling
- DNA profiling is about the differences in introns as most humans have very similar exons
- introns are variable between people as the aren’t expressed so make no difference to the organism
- a common difference is the number of VNTR’s or variable number tandem repeats
- mutations that affect the length of introns are: addition, deletion, duplication and translocation (one intron to another)
- mutations that do not affect the length of introns are substitution and inversion
