8B: Genome Flashcards
genome
complete set of genes
proteome
full range of proteins a cell can produce
Whole genome shotgun sequencing
involves researchers cutting the DNA into small, easily sequenced sections and then using computer algorithms to align overlapping segments to assemble the genome.
Why is determining the genome and proteome of bacteria relatively easy?
- Most prokaryotes have just one circular piece of DNA not associated with histones
- There are none of the non-coding sections of DNA (introns)
Why is determining the genome and proteome of complex organisms harder?
- The DNA contains large sections of non-coding DNA (introns)
- Contain complex regulatory genes that determine when the genes that code for a particular protein are switched on and off
What does recombinant DNA technology involve?
Involves the transfer of fragments of DNA from one organism or species to another
What is recombinant DNA?
where fragments of foreign DNA are inserted into other sections of DNA.
What is the name of the recipient of the recombinant DNA?
transgenic
How is it possible that the DNA of one organism is not only accepted by a different species but also functions normally?
- The genetic code is universal
- Mechanisms of transcription and translation are essentially the same in all living organisms
Process of making a protein using DNA technology of gene transfer and cloning
1) Isolation of the DNA fragments that have the gene for the desired protein
. Using reverse transcriptase
. Using restriction endonuclease
enzymes
. Using a gene machine
2) Insertion of the DNA fragment into a vector
3) Transformation -> the transfer of DNA into suitable host cells
4) Identification of host cells that have successfully taken up gene using gene markers
5) Amplification
Isolation of the DNA fragments that have the gene for the desired protein
Process:
1) A cell that naturally produces the protein of interest is selected
2) These cells have large amounts of mRNA for the protein, so it is more easily extracted.
3) mRNA acts as a template for the reverse transcriptase enzyme
4) This joins free DNA nucleotides with complementary bases to the mRNA sequence.
5) Single-stranded cDNA is isolated by hydrolysis of the mRNA with an enzyme
6) Double-stranded DNA is then formed on the cDNA template using the enzyme DNA polymerase.
7) This double strand of DNA is the required gene
8) The cDNA is intron-free because it is based on the mRNA template.
Using restriction endonuclease enzymes
Process:
1) Each restriction endonuclease cuts the DNA at a recognition sequence where it has an active site which is complementary to the recognition sequence
2) The DNA is incubated with the specific restriction endonuclease
3) The restriction enzyme hydrolyses the DNA, leaving sticky ends
Why are sticky ends important?
- The cut ends of the DNA double strand are left with a single strand, which is a few bases long
- they can anneal the DNA fragments with other pieces of DNA with complementary base pairs
Using a gene machine
Synthesis of DNA fragments from scratch without the need for a pre-existing DNA template using a computerised machine
What are the advantages of using mRNA to make a DNA fragment
- more mRNA in cell than DNA so it is easily extracted
- introns removed by splicing whereas DNA contains introns
- bacteria can’t remove introns
What are the advantages using a gene machine?
- any sequence of nucleotides can be produced
- short time
- good accuracy
What are the 2 ways of amplifying fragments of DNA?
1) In vivo - copies are made inside a living organism by transferring the fragments to a host cell using a vector
2) In vitro - copies of the DNA fragment are made outside the living organism using PCR
What are primers?
short sequences of nucleotides that have a set of bases complementary to those at the end of each of the 2 DNA fragments
PCR Stage 1:
1) A mixture of the DNA fragments, primers, and DNA polymerase is placed in a vessel with a thermocycler
2) Temperature is increased to 95 degrees Celsius, causing the 2 strands of the DNA fragments to separate due to breaking of hydrogen bonds
PCR- Stage 2
3) The mixture is cooled to 55 degrees Celsius, causing the primers to anneal to their complementary base at the end of the DNA fragment
4) The primers provide the starting sequences for DNA polymerase to begin DNA copying because DNA Taq polymerase can only attach nucleotides to the end of an existing chain.
5) Primers also prevent the two separate strands from simply rejoining.
6) Need 2 different primers are required (forward and reverse) because DNA strands run in anti-parallel, but polymerase can only run in one direction
PCR- Stage 3
7) The temperature is increased to 72 °C so DNA polymerase can work at optimum temperature
8) DNA taq polymerase adds free complementary nucleotides along each of the separated DNA template strands
9) It begins at the primer on both strands and adds the nucleotides in sequence until it reaches the end of the chain.
10) By complementary base pairing, new complementary strands are formed
11) Because both separated strands are copied simultaneously there are now two copies of the original fragment
12) Once the 2 DNA strands are completed, the process is repeated by subjecting them to the temperature cycle again, resulting in four strands.
13) Every cycle doubles the DNA
Advantages of in vivo
- useful in recombinant DNA
- almost no risk of contamination
- very accurate
- very precise
Advantages of in vitro
- extremely rapid
- does not require living cells
