Recombinant DNA technology

Question 1

polymerase chain reaction:

Answer 1

a method of copying fragments of DNA, this process is automated, making it both rapid and efficient

Question 2

process of PCR

Answer 2

• DNA FRAGMENT: to be copied
• DNA POLYMERASE: enzyme capable of joining together thousands in a matter of minutes. an enzyme taq polymerase is obtained from bacteria in hot springs and is tolerable to heat so it doesn’t denature during high temps
• PRIMERS: short sequences of nucleotides that have a set of bases complementry to those at one end of the 2 DNA fragments
• NUCLEOTIDES: which contain each of the four bases found in DNA
• THERMOCYCLER: a computer controlled machine that varies in temperature

Question 3

Polymerase chain reaction and is carried out in 3 stages

Answer 3

• separation of the DNA strand: the dna fragments, primers and dna polymerase are placed in the thermocyler and the temp is increased to 95 degrees causing the dna fragments to separate due to the breaking of the hydrogen bonds
• annealing of the primers: mixture is cooled to 55/40 degrees causing the primers to join to their complementary bases at the end of the DNA fragment. primers provide the starting sequence for DNA polymerase to begin. they also prevent the two separate strands from rejoining
• synthesis of DNA: temperature is increased to 72 degrees. this is the optimum temp for the DNA polymerase to add complementary nucleotides along each of the separated DNA strands. it begins at the primer on both strands and adds nucleotides until it reaches the end of the chain

Question 4

Advantages of in vitro gene cloning:

Answer 4

• extremely rapid: within a matter of hours a 100 billion copies of a gene can be made. this can be valuable to a crime scene where a minute amount of DNA is avaliable and this can be increased using the PCR so no valuable time is lost before forensic analysis
• It doesn’t require living cells: all that is required is a base sequence of DNA that needs amplification. No complex culturing techniques, requiring time and effort are needed

Question 5

In vitro

Answer 5

in vitro uses the polymerase chain reaction

Question 6

in vivo

Answer 6

in vivo transfers the fragments to a host cell using a vector

Question 7

importance of sticky ends

Answer 7

• recognition sites: where DNA is cut by restriction endonucleases
• if recognition site is cut in a staggered fashion, the ends of the DNA double strand which is a few nucleotide bases long
• the nucleotides on a single strand at one side of the cut are complementary to those at the other side
• the single-stranded end of any one fragment can be stuck to the single ended strand of any other fragment, their ends are sticky
• once the complementary bases of 2 sticky ends have paired, DNA ligase is used to bind the phosphate-sugar framework of the two sections of DNA
• sticky ends are important because provided the same restriction endonuclease is used, the DNA of one organism can be combined

Question 8

what is recombinant DNA technology

Answer 8

technology that allows genes to be manipulated, altered and transferred from organism to organism

Question 9

The process of making a protein using the DNA technology of gene transfer and cloning involves a number of stages:

Answer 9

1) isolation of the DNA fragments that have the gene for the desired protein
2) insertion: of the DNA fragment into a vector
3) transformation that is the transfer of DNA into suitable host cells
4) identification of the host cells that have successfully taken up the gene by use of gene markers
5) growth/cloning of the population of host cells

Question 10

there are several methods of producing DNA fragments

Answer 10

• conversion of mRNA to cDNA using reverse transcriptase
• using restriction endonucleases to cut fragments containing the desired gene from DNA
• creating the gene in a gene machine, usually based on protein structure

Question 11

using reverse transcriptase:

Answer 11

retroviruses