Genome Projects and Gene Technology - A16 Flashcards
What is a genome?
complete set of genes in an organism
What is a proteome?
The range of proteins an organism can produce
Describe the process of sequencing proteomes of simple organisms.
-bacteria do not have non-coding DNA
-easy to determine their proteome from their DNA sequence of their genome
-useful in medical research - identifies protein antigens on the surface of disease causing bacteria and viruses - vaccines
-also allow scientists to monitor pathogens during an outbreak of a disease - leads to better management of the spread of infection and can help identify antibiotic resistant factors
Describe the process of sequencing proteomes of complex organisms
-contain large sections of non-coding DNA
-contain complex regulatory genes, which determine when the genes that code for a particular protein are turned on and off
-both of these make it difficult to translate a genome into a proteome because it’s hard to find the parts of the genome that code for proteins
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-in the past, sequencing methods were labour-intensive, expensive and could only be done on small scale
-now, techniques are automated, more cost effective and can be done on a large scale
-with newer, faster techniques such as pyrosequencing available, scientists can now sequence whole genomes much more quickly
What does recombinant DNA technology involve?
-transferring a fragment of DNA from one organisms to another
-because the genetic code is universal, and because transcription and translation mechanisms are universal, the transferred DNA can be used to produce proteins in the cells of the recipient organisms
-donor and recipient organisms do not have to be the same species
What are transgenic organisms?
organisms that contain transferred DNA
What are the different methods for gene fragments?
-to transfer a gene from one organism to another, you need to get a DNA fragment containing the gene you are interested in(target gene)
1)using reverse transcriptase
2)using restriction endonuclease enzymes
3)using a gene machine
How is reverse transcriptase used to obtain gene fragments?
-most cells only contain two copies of each gene, making it difficult to obtain a DNA fragment containing the target gene.
-but cells that produce the protein coded for by the target gene will contain many mRNA molecules which are complementary to the gene-so mRNA is easier to obtain
1)reverse transcriptase makes DNA from an RNA template
2)the DNA produced is called complementary DNA(cDNA)
3)mRNA must first be isolated, then mixed with free DNA nucleotides and reverse transcriptase, making cDNA
How are restrictions endonuclease enzymes used to obtain gene fragments?
-some sections of DNA have palindromic sequences of nucleotides
-these sequences consist of anti parallel base pairs(base pairs that read the same in the opposite direction)
-restriction endonucleases are enzymes that recognise specific palindromic sequences(known as recognition sites) and cut(digest) the DNA at these places.
-different restriction endonucleases cut different specific recognition sequences because the recognition site is complementary to the enzymes active site
-if recognition sites are present at either side of the DNA fragment you want, you can use restriction endonucleases to cut the fragment out from the rest of the DNA.
-the DNA sample is incubated with the specific restriction endonucleases, which cuts the DNA fragment out via a hydrolysis reaction
-sometimes the cut leaves sticky ends - small tails of unpaired bases at each end of the fragment
-sticky ends can be used to bind(anneal) the DNA fragment to another piece of DNA that has complementary sticky ends
How is a gene machine used to obtain gene fragments?
-recently technology has developed so that fragments of DNA can be synthesised from scratch, without the need for pre-existing DNA template-made from the knowledge of the primary sequence
-instead, a database contains all the necessary information to produce the DNA fragment
-this means that the DNA sequence does not have to exist naturally- any sequence can be made
1)the sequence is designed
2)the first nucleotide in the sequence is fixed to a support e.g. a bead
3)nucleotides are added step by step the correct order, in a cycle of processes that includes adding protecting groups. Protecting groups make sure the nucleotides are joined at the right points, to prevent unwanted branching
4)shirt sections if DNA called oligonucleotides, rouggky 20 nucleotides long, are produced. Once these are complete, they are broken off from the suppor and all the protecting groups are removed. The oligonucleotides can then be joined together to make longer DNA fragments
What is in vivo cloning?
Gene copes ade made within a living organism. As the living organism grows and divides, it replicates the DNA making many copes of the gene.
What is in vitro cloning?
Gene copies are made outside a living organism using polymerase chain reaction(PCR)
What is a vector?
Something used ti transfer DNA into a cell - plasmids or bacteriophages
Describe the steps in part one of in vivo cloning.
-first step is to insert the DNA fragment in a vector’s DNA
-the vector is isolated and restriction endonucleases and DNA kinase are used to stick the DNA fragment and vector DNA together
1)vector DNA is isolated
2)vector DNA is cut open using the same restriction endonucleases that was used to isolate the target gene. Causes sticky ends or the vector and target gene to be comllementary
3)vector DNA and DNA fragment are mixed with DNA ligase. Ligase joins the sticky ends of the DNA fragment and vector DNA- ligation, by complementary base pairing
4)vector DNA+DNA fragment= recombinant DNA
What is a bacteriophage?
Infect the host bacteria and inject its DNA, which is then integrated into the bacterial DNA
Describe the steps in part two of in vivo cloning.
-recombinant DNA transfers the target gene into the host cells
-host cells that take up the vector containing the target gene are said to be transformed
-if a plasmid is used, host cells gave to be persuaded to take in recombinant plasmid
Describe the steps in part three of in vivo cloning.
-only about 5% of host cells will take up the recombinant DNA si we must identify which cells have been transformed
-marker genes are used to identify these cells
1)marker genes are inserted into the vectors at the same time as the target gene. All transformed host cells will therefore contain the marker and the target gene.
2)host cells are gorwn on agar plates and each cell duivides and replicates its DNA, producing a colony of cloned cells.
The marker gene can code for antibiotic resistance - host cells grown on agar plates containing a soecific antibiotic, so only transformed cells have the marker gene will survive and grow. Marker gene can also code for fluorescence
3) identified transformed cells are allowed to grow more, producing many copies of the cloned gene.
What is a promotor region?
DNA sequences that tell RNA polymerase where to start producing mRNA
What is the terminator region?
Tell RNA polymerase where to stop producing mRNA
How do transformed host cells produce proteins?
-for trabsformed host cells to produce the protein coded for by the target gene, the vector must contain specific promoter and terminator regions.
-without these regions, a DNA fragment won’t be transcribed and translated
-promoter and terminator regions may be present in the vector or added in along with the DNA fragment
What is in vitro cloning?
-DNA fragments can be amplified in vitro -DNA copies made outside of a living organisms using polymerase chain reaction(PCR)
-PCR makes millions o copies of DNA fragments in just a few hours
-PCR has several stages that repeated many time to make many copies
What is a primer?
short pieces of single stranded DNA that are complementary to the bases at the start of the fragment you want.
What is DNA polymerase?
an enzyme that joins DNA nucleotides to form DNA.
Uses phosphodiester bonds
