Recombinant DNA Technology

Question 1

how is reverse transcriptase used to isolate fragments of DNA?

Answer 1

• retroviruses contain reverse transcriptase enzyme
• catalyses formation of a double strand of DNA from a single strand of RNA
• single stranded DNA polypeptide made which is complementary to RNA
• then converted into a double-stranded DNA molecule called cDNA by DNA polymerase

Question 2

how do restriction endonucleases cut fragments of DNA?

Answer 2

• enzymes extracted from bacteria that cut DNA at specific base sequences (recognition sites)
• sequences are often 4-8 base pairs long

Question 3

what is the importance of sticky ends?

Answer 3

• if the same restriction endonuclease is used to cut 2 DNA fragments then the ends will be complimentary
• the fragments of DNA cut by the same restriction endonuclease will be able to join together (allow DNA from different organisms to join together and form recombinant DNA)

Question 4

how can a DNA fragment be inserted into a vector?

Answer 4

• plasmid and gene are cut with the same restriction enzyme to create complimentary sticky ends
• the inserted DNA and vector are complementary and can be joined
• fragments are incubated with the plasmids, base pairing will take place between complementary sticky ends which are sealed with DNA ligase which forms phosphodiester linkages
• a recombinant DNA molecule is created

Question 5

how is the DNA of the vector introduced into host cells?

Answer 5

• plasmids are introduced into bacterial cells by transformation
• electroporation is used to stimulate bacterial cells to take up plasmids
• electroporation facilities this process by increasing the permeability of bacterial membranes thus increasing chances of success
• uses calcium salts and rapid temperature change from 0 to 40 degrees

Question 6

what are gene markers and how do they work?

Answer 6

• used to see whether the DNA has been taken up by the bacteria
• different types of gene markers are: antibiotic resistant genes, fluorescent markers and enzyme markers
• these genes are incorporated into the plasmid, so that bacteria with the plasmid can be separated from those that don’t have the plasmid
• they can also determine whether the desired DNA has entered the plasmid, as the marker gene will become inactivated

Question 7

what is the polymerase chain reaction?

Answer 7

• amplifies DNA by making millions of copies of a DNA sample

Question 8

how is the PCR carried out?

Answer 8

• a reaction mixture is set up including the DNA sample, primers, free nucleotides and DNA polymerase
• the mixture is heated to 95 degrees to break the hydrogen bonds and separate the strands
• the mixture is cooled to around 50-65 and the primers anneal to the strands
• temperature increases to 70, as this is the temperature that DNA polymerase works at - the DNA polymerase used is Taq polymerase and comes from bacteria that live in hot springs
• DNA polymerase creates a copy of the sample by complementary base pairing using free nucleotides
• the cycle is repeated around 30 times and this gives a sufficient amount of DNA to create a DNA profile

Question 9

what are the advantages and disadvantages of in-vitro cloning?

Answer 9

• in vitro = in glass/lab
• can be done with PCR
• fast, automated and reliable
• doesn’t require living cells
• can have problems such as contamination and errors

Question 10

what are the advantages and disadvantages of in-vivo cloning?

Answer 10

• in vivo = in life
• cane done using recombinant plasmids in bacterial cells
• accurate and useful as the gene is placed in cells where it can be expressed
• very time consuming
• requires monitoring of cell growth

Question 11

what is a DNA probe?

Answer 11

• short, single stranded DNA molecule
• designed to be complementary to base sequence to be detected
• made in smaller quantities and amplified
• uses either radioactive isotopes or a fluorescent dye which glows under certain wavelengths of light

Question 12

what is the process of using DNA probes?

Answer 12

• sequence of nucleotides on mutated gene is determined by DNA sequencing
• fragment of DNA with complementary bases is produced
• DNA probe formed by fluorescently labelling the DNA fragment
• PCR produces many copies
• probe is added to single stranded DNA fragments
• if the donor has the mutated gene, the probe will bind to the complementary bases on the DNA fragment
• the fragments are now labelled with the probe

Question 13

what is genetic fingerprinting used for?

Answer 13

• detect differences in peoples DNA

Question 14

how does DNA fingerprinting work?

Answer 14

• uses variable number tandem repeats (VNTRs) which are short repeating sequences or bases in non-coding regions of DNA
• the probability of 2 individuals having identical VNTRs is extremely low

Question 15

what is gel electrophoresis?

Answer 15

• process used to separate the DNA fragments according to their size using an electric current

Question 16

what can genetic engineering be used for?

Answer 16

• forensic science
• medical diagnosis (source of infection)
• plant and animal breeding (breeding programmes - prevent inbreeding)

Question 17

what is the process of genetic fingerprinting?

Answer 17

• DNA is extracted from the sample
• restriction endonuclease cut the DNA into fragments
• fragments are separated using gel electrophoresis and transferred from gel to nylon membrane
• DNA probes are added to label the fragments in DNA hybridisation
• membrane with radioactively labeled DNA fragments is placed onto an X-ray film
• development of the X-ray film reveals dark bands where radioactive DNA probes have attached

Question 18

what is DNA hybridisation?

Answer 18

• when a section of DNA or RNA is combined with a single stranded section of DNA which has complementary base
• before hybridisation can occur, the two strands of DNA must be separated (denaturalisation)
• when this occurs the complementary bases on each strand recombine (anneal)