Recombinant DNA Technology

Question 1

give a summary of what recombinant dna technology is

Answer 1

it is the joining together of dna molecules from different organisms and inserting it into a host to produce new genetic combinations
dna from different organisms is cut and pasted together, producing recombinant dna

Question 2

what is the difference between our dna and plasmid dna

Answer 2

both dna are double stranded molecules and the nucleotides use bases to form the hydrogen bonds with the bases on the opposing strand to form a double helix. however, our dna has 46 chromosomes and plasmid dna is circular

Question 3

what does dna cloning involve

Answer 3

digesting dna that contains the target gene we want to clone. this digesting is done by restriction enzymes such as EcoR1 and they bind to specific nucleotide sequences called restriction sites

Question 4

what is inserted into the plasmid alongside the target gene

Answer 4

a gene for antibiotic resistance so we can selectively grow only bacteria with recombinant plasmid

Question 5

what is recombinant dna technology valuable for

Answer 5

science, medicine, agriculture and industry

Question 6

where is recombinant dna technology important for medicine

Answer 6

• vaccine production
• protein therapies (human insulin, human growth hormone, inferferon)
• production of blood clotting factrors to treat haemophilia
• gene is cloned into a plasmid which is then introduced into a bacterial cell. the bacteria produce the protein which is then purified and used in patients.

Question 7

what are some other applications of recombinant dna technology

Answer 7

gene therapy and in transgenic animals

Question 8

how is recombinant dna technology used in gene therapy

Answer 8

they replace faulty mutated genes with healthy ones or add a new gene into the genome. this can be used to treat or prevent disease like cancer, diabetes, heart disease, cistic fibrosis and haemophilia

Question 9

how is recombinant dna technology used in transgenic animals

Answer 9

possess an integrated gene or dna sequence in the genome which can be passed onto offspring. can lead to improved reproductive performance, increased growth rate, improved carcass composition, improved milk production and quality, and increased disease resistance

Question 10

who is dolly the sheep

Answer 10

this was the first mammal to be cloned from an adult somatic cell using the process of nuclear transfer. she was born in july 1996 at the roslin institute and died february 2003 from lung cancer. she is now on display at the national museum of scotland

Question 11

give an overview of gene cloning

Answer 11

this produces a large number of copies of a particular piece of dna. the genes are usually cloned by isolating them using restriction enzymmes, followed by gel electrophoresis and then inserting them into a plasmid. the plasmid is then introduced into a bacterium, and the bacterium is allowed to grow to produce large numebrs of cells and hence many copies of the same gene. the gene can then be reisolated using the same restriction enzyme

Question 12

what are restriction enzymes

Answer 12

these are enzymes that cut double stranded dna at specific dna sequences.
they typically are 4-6 base pairs in length and are palindromic, which means they read the same in both directions.
most restriction enzymes make a staggered cut which forms sticky ends.
sticky ends allow dna fragments to re associate by base pairing.
after reassociation, the fragments can be rejoined by dna ligase.

Question 13

when were restriction enzymes discovered

Answer 13

1971

Question 14

what are some examples of restriction enzymes that leave sticky ends

Answer 14

EcoR1 and Msp1

Question 15

what are examples of restriction enzymes that leave flush ends

Answer 15

HaeIII, EcorV, Bal1

Question 16

describe the breakdown of the action of EcoRI

Answer 16

the enzyme cuts both dna strands at the same time.
the dna fragments of foreign dna with complementary sequence the cut DNA join at sticky ends.
dna ligase enzyme joins the two together.
this forms recombinant dna

Question 17

explain the process of gel electrophoresis

Answer 17

it is used to separate dna fragments on the basis of their size
samples are applied to a gel immersed in a bugger and a current is applied.
negatively charged dna migrates from the negative electrode to the positive electrode. the larger dna fragments migrate more slowly than smaller dna fragments, so they can be separated according to their size

Question 18

breakdown the gene cloning process please

Answer 18

• to insert a gene into a plasmid, a restriction enzyme is chosen that cuts on either side of the gene but not in the middle - so the gene is contained on a single dna fragment
• the gene is separated from other dna fragments by gel electrophoresis - so we know the size and isolate and purify it
• a suitable plasmid is linearised (cut at one point) using the same restriction enzyme - plasmids are engineered to have a number of restriction sites
• the cut plasmid and gene are mixed, and the sticky ends (sticky end cloning is more efficient than blunt end cloning) of the plasmid and gene are allowed to anneal (associated by base pairing) .
• the annealed ends are covalently joined using dna ligase
• the plasmid, now containing the gene of interest, is introduced into the host bacterium
• the bacteria are grown into a colony, using antibiotic resistance genes in the plasmid to select colonies containing plasmids
• cloned cells are lysed and the plasmids isolated by centrifugation
• plasmids are cut with restriction enzyme, releasing the cloned gene
• plasmids are circular pieces of dna that have been engineered to allow genes to be cloned into it

Question 19

how is antibiotic resistance involved in gene cloning

Answer 19

plasmids all have at least two antibiotic resistant genes. they allow us to select e coli cells that have plasmids that allow cloning. because gene cloning isnt 100% efficient, we need to be able to select the e coli cells that have the plasmid with a successfully cloned gene. this is done through antibiotic resistance conferred onto the plasmid that has been successfully cloned. foreign dna is inserted into the plasmid for resistance. if cloning is successful, the foreign dna has been inserted into this gene and is disrupting the resistance, which leaves the remaining undisruptant gene

Question 20

how would you know if the plasmid did not take up the foreign dna

Answer 20

it would not be antibiotic resistant

Question 21

what is dna sequencing used for

Answer 21

determining base sequences of dna. it works out the structure of a gene or an entire genome

Question 22

what is sangar sequencing

Answer 22

this is the dideoxynucleotide chain termination method. it involves the synthesis of new dna strands complementary to a single stranded template strand in vitro

Question 23

what are some reaction components of the manual approach in sangar sequencing

Answer 23

• a single stranded dna template
• primer
• deoxynucleotides
• dideoxynucleotides
• dna polymerase
• label

Question 24

what is the single stranded dna template in sangar sequencing used for

Answer 24

because its sequence is to be determined, it is used as a template for the synthesis of a complementary strand

Question 25

what is the primer and how is it used during sangar sequencing

Answer 25

short oligonucleotide that serves as a primer for the synthesis of the complementary dna strand by primer extension

Question 26

how are deoxynucleotides used during sangar sequencing

Answer 26

they are the building blocks of DNA

Question 27

what are the deoxynucleotides

Answer 27

dATP, dCTP, dGTP, dTTP

Question 28

what are the dideoxynucleotides used for in sangar sequencing

Answer 28

these are modified nucleotides that terminate the dna strand elongation

Question 29

what are the dideoxynucleotides

Answer 29

ddATp, ddCTP, ddGTP, ddTTP

Question 30

how is dna polymerase used in sangar sequencing

Answer 30

it is the enzyme that catalyses dna strand synthesis

Question 31

what are the labels used in sangar sequencing and how do they aid the process

Answer 31

fluorescent or radioactive labels are used, and they are required to visualise the products. the label primer i son the 5 prime end.

Question 32

what is chain terminationwhat does interruption of the dna strand synthesis depend on

Answer 32

the presence of ddNTPs

Question 33

how do ddNTPs terminate dna strand elongation

Answer 33

they do this by interrupting the dna strand synthesis. the 3 prime hydroxyl groups of the dNTPs are replaced by the hydrogen ion in the corresponding ddNTP. this incorporates the ddNTP into the growing dna chain as the final nucleotide, and because it lacks the 3prime hydroxyl group required to form a phosphodiester bone with the next nucleotide, there is chain termination.

Question 34

why are there hundreds of dna molecules of varying length

Answer 34

because dna synthesis can be interrupted at every possible site in a given population of molecules

Question 35

how can it be ensured that the dna strands are still allowed to elongate sufficiently for the sequence analysis

Answer 35

the ddNTPs are added at much lower concentrations than the standard dNTPs

Question 36

give a breakdown on the procedure for dna sequencing

Answer 36

the dna to be sequenced is mixed with primer. the primer binds to the 3 prime end of dna.
this mixture is divided into four separate reaction tubes containing all four dNTPs, one of the four ddNTPs and dna polymerase.
chain synthesis proceeds in each of the four reaction mixtures
gel electrophoresis separation of reaction products leads to the band corresponding to each position of the chain termination to appear.
dna bands are detected by autoradiography or by a laser in an automated sequencer. the dna sequence can be deduced from the pattern of bands in the four lanes

Question 37

what are the possible patterns of bands that can be viewed in the four lanes on gel electropheresis following dna sequencing

Answer 37

• a dark band, which indicates a dna fragement that is the result of chain termination after incorporating a ddNTP
• a terminal nucleotide base, which can be identified according to which ddNTP was added in the reaction giving that band
• the relative positions of the different bands among the four lanes are then used to read from the bottom to the top of the dna sequence

Question 38

what is the automated approach to dna sequencing

Answer 38

this is dye terminator sequencing, and involves smaller fragments passing through capillaries first.
each band of colour is caused by the collections of dye terminated fragements of the same size. as each band of colour moves past the detector, it creates a peak in the signal which is produced on a graph.

Question 39

compare the efficiency of gel based vs automated sanger sequencing throughput

Answer 39

• gel based generates only 250-500 base pairs of sequence per sample, and 8 samples can be sequenced per run. it is more hands on, and time inefficient
• automated generates 740-1000 base pairs of sequence per sample and up to 96 samples can be sequenced per run. it is more time efficient

Question 40

what is high throughput sequencing

Answer 40

this is also known as next generation sequencing, and there is no need for cloning as it is highly scalable.
it involves the sequencing of millions of genes and entire genomes at once.
it is cheap and rapid, and requires substantial bioinformatics analysis
many platforms are illumina and roche.

Question 41

which enzymes produce blunt ends

Answer 41

Hae III
EcoR V
Bal I

Question 42

which enzymes produce sticky ends

Answer 42

EcoR I
Msp I