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Question 1

What is recombinant DNA technology

Answer 1

Joining together of DNA molecules from different organisms and inserting it into a host to produce new genetic combinations of value to science, medicine, agriculture and industry

Question 2

What does recombinant DNA tech involve

Answer 2

DNA from different organisms is “cut and pasted” together, producing recombinant DNA

Question 3

When was the first recombinant DNA molecule produced

Answer 3

1972 by Paul Berg who won a Nobel prize for chemistry in 1980

Question 4

Why is recombinant DNA tech important

Answer 4

Vaccine production

Protein therapies
-insulin
-growth hormones
-interferon

Production of blood clotting factors to treat haemophilia

To produce these proteins the gene is cloned into a plasmid which is then introduced into a bacterial cell; bacteria produce the protein, which is then purified and used in patients

Question 5

What is gene therapy

Answer 5

-Replace faulty (mutated) gene with a healthy one and add a new gene into the genome
-Used to treat or prevent disease e.g. cancer diabetes, heart disease, cystic fibrosis, haemophilia

Question 6

How can DNA tech be used with transgenic animals

Answer 6

-They possess an integrated gene or DNA sequence (transgene) in the genome, which can be passed onto offspring
-Improved reproductive performance, increased growth rate, improved carcass composition, improved milk production and/or quality, increased disease resistance

Question 7

What was the first mammal to be cloned

Answer 7

Dolly the sheep

Question 8

What is gene cloning

Answer 8

> It produces large numbers of copies of a particular piece of DNA
Genes are usually cloned by isolating them using restriction enzymes, followed by gel electrophoresis and inserting them into a plasmid
The plasmid is then introduced into a bacterium, and the bacterium allowed to grow to produce large numbers of cells and hence many copies of the gene
The gene can then be re-isolated using the same restriction enzyme

Question 9

What do restriction enzymes do

Answer 9

Cut double-stranded DNA at specific DNA sequences

EX
Blunt - Hae III
Sticky - EcoR V MORE EFFICIENT

Question 10

What is Gel electrophoresis

Answer 10

-Used to separate DNA fragments on the basis of their size
-Samples are applied to a gel immersed in buffer and a current is applied
-Negatively-charged DNA migrates from the negative electrode (top of gel) to the positive electrode (bottom of gel)
-Larger DNA fragments migrate more slowly than smaller DNA fragments allowing separation by size

Question 11

Process of gene cloning

Answer 11

1. To insert a gene into a plasmid, a restriction enzyme is chosen that cuts on either side of the gene but not the middle
2. The gene is separated from other DNA fragments by gel electrophoresis
3. A suitable plasmid is cut at one point using the same restriction enzyme
4. The cut plasmid and gene are mixed, and the sticky ends of the plasmid and gene are allowed to anneal by base pairing
5. The annealed ends are covalently joined using DNA ligase
6. The plasmid, now containing the gene of interest, is introduced into the host bacterium
7. The bacteria are grown into a colony, using antibiotic resistance genes in the plasmid to select colonies containing plasmids
8. Cloned cells are lysed and the plasmids isolated by centrifugation
9. Plasmids are cut with the restriction enzyme, releasing the cloned gene for further studying

Question 12

Why do we use DNA sequencing

Answer 12

To determine base sequences of DNA
Works out the structure of a gene or an entire genome e.g. human genome project

Question 13

What is sanger sequencing also known as

Answer 13

Dideoxynucleoside chain termination method (used for small scale projects)

Answer 14

Synthesise new DNA strands which are complementary to a single stranded template strand in vitro (primer extension)

500 base pair stretch - generates 500 different DNA molecules in vitro

Question 15

What are the components in the manual approach to sanger sequencing

Answer 15

1. Single stranded DNA template - unknown sequence used as a template for the synthesis of complementary strand
2. Primer -short oligonucleotide serves as a primer from synthesis of comp DNA strand
3. dNTPs (deoxynucleotides): dATP dCTP dGTP dTTP (building blocks of DNA)
4. ddNTPs (dideoxynucleotides): ddATP ddCTP ddGTP ddTTP (modified nucleotides that terminate DNA strand elongation
5. DNA polymerase - enzyme that catalyses DNA strand synthesis
6. Label - Fluorescent (IRD800) or radioactive (35S). Required to visualise products. Label primer at 5 prime end

Question 16

How does chain termination occur

Answer 16

> Interruption of DNA strand synthesis depends on presence of ddNTPs
The 3’ - OH group in dNTPs is replaced by -H in ddNTPs
ddNTPs are incorporated into growing DNA chain but as they lack the 3’ - OH required to form a phosphodiester bond with the nest nucleotide - Chain termination occurs
Synthesis is interrupted at every possible site in a given population of molecules, resulting in hundreds of DNA fragments of varying length
ddNTPs are added at a much lower concentration than the standard dNTPs to allow strand elongation sufficient for sequence analysis

Question 17

Laboratory procedure of DNA sequencing using sanger method

Answer 17

1. DNA to be sequences is mixed with primer
2. Primer binds to 3’ end of DNA
3. DNA-primer mixture is divided into four separate reaction tubes containing:
   -all four dNTPs
   -one of the four ddNTPs
   -DNA polymerase
4. Chain synthesis proceeds in each of the four reaction mixtures
5. Gel electrophoresis (highly resolving) separation of reaction products - bands corresponding to each position of chain termination appears
6. DNA bands detected by autoradiography or by laser in an automated sequencer
7. DNA sequence can be deduced from the pattern of bands in the four lanes:
   -a dark band in a lane indicates a DNA fragment that is the results of chain termination after incorporation of a ddNTP
   -the terminal nucleotide base can be identified according to which ddNTP was added in the reaction producing that band
   -the relative positions of the different bands among the four lanes are then used to read (from bottom to top) the DNA sequence

Question 18

What enzyme catalysis chain synthesis

Answer 18

DNA polymerase

Question 19

What is the name for the automated approach of sequencing

Answer 19

Dye terminator sequencing
-each terminal end is coloured corresponding to the base and produce peaks example:
A- blue
C- blue
G- yellow
T- red

Question 20

Differences between Gel-based and automated sanger sequencing

Answer 20

GEL BASED
-250 to 500 base pairs of sequence per sample
-Up to 8 samples sequenced per run

AUTOMATED (more efficient)
-750 to 1000 base pairs of sequence per sample
-Up to 96 samples sequenced for run

Question 21

Alternative name for high throughput sequencing

Answer 21

Next Generation Sequencing

Question 22

Advantages of high throughput (NGS) sequencing

Answer 22

-No need for cloning, highly scalable
-Sequence millions of genes and entire genomes at once
-Cheap and rapid
-Requires substantial bioinformatics analysis

Question 23

What are the four main DNA sequencing methods used by NGS systems

Answer 23

Pyrosequencing
Sequencing by synthesis
Sequencing by ligation
Ion semiconductor sequencing

Question 24

Why has the cost of human genome sequencing decreased

Answer 24

Due to the development of sequencing from sanger (manual) to NGS