Genetic Tools

Question 1

What is PCR?

Answer 1

a test for the amplification of DNA

Question 2

what are the 3 steps in PCR?

Answer 2

1. Denaturation
   double DNA strands melts open
2. Annealing
   primers bind to DNA, polymerase starts copying DNA
3. Extension
   at 72oC

Question 3

repetition of pcr

Answer 3

heat and then cool
allows primers to bind to DNA
cycles repeat
30-40 cycles

Question 4

Amplification

Answer 4

Only DNA between primers is amplified
ends are identified by the primers added

Question 5

What are the PCR primers?

Answer 5

short
20bp
single stranded DNA
chemically synthesised

Question 6

what is agarose gel electrophoresis?

Answer 6

lab technique
provides matrix for DNA
speed varies depends on size and shape of the product
lighter move faster than heavier

Question 7

separation of DNA by size

Answer 7

Potential difference along the gel
moves to positive electrode
dependent on conformation & size

stain DNA with fluorescent dye for detection by UV exposure

Question 8

What is Sanger Sequencing?

Answer 8

Mix of different length DNA strands
each terminating with fluorescent nucleotide
run through capillary tube & at end there is a laser reading
smallest first then larger will read

Question 9

What are the uses of PCR?

Answer 9

detection of pathogens in water
dna sequencing
genetic fingerprinting
forensic analysis
diagnosis of genetic disorders

Question 10

What are the limitations of PCR?

Answer 10

sequence information required
limit on length
potentially high error rate
very sensitive to exact reaction conditions
need primers so need background info

Question 11

Key points of PCR

Answer 11

use for in vitro DNA synthesis
requires prior knowledge
gel electrophoresis used to visualise
broadly used lab technique

Question 12

what are plasmids?

Answer 12

plasmids are small extra chromsomal dna
& can be shared between bacteria

Question 13

How do you cut DNA?

Answer 13

by restriction endonucelases
creating 5’ overhang

Question 14

How do you join DNA?

Answer 14

DNA ligase
forming phosphodiester bonds
requiring ATP

Question 15

What is recombinant DNA?

Answer 15

a set of dna that is within a cloning vector
1. cloning vector cleaved by restriction nuclease
2. covalent linkage of DNA fragment by DNA ligase
3. creating of recombinant DNA

Question 16

What is gene cloning?

Answer 16

introduction of recombinant plasmid into bacterial cell
replication and cell division

can recover the DNA for analysis

can also clone genes by insertion into bacteriophage

Question 17

Collection of recombinant clones

Answer 17

canoe frozen and stored
creating a gene library for future use

Question 18

What is transgenics?

Answer 18

genes between species
the genetic code is universal between organisms

Question 19

How can DNA be expressed in cells?

Answer 19

insert promoter region to create protein of interest
remove an introns present- CDNA

make a CDNA copy by removing introns from mRNA

1. introduction of DNA into cell
2. replication and expression in the cell

Question 20

How do you make CDNA?

Answer 20

1. Extract RNA
2. Reverse transcribe
3. Treat with RNAse H
4. Make second strand of DNA with DNA polymerase

Question 21

What are examples of genetic engineering?

Answer 21

human insulin
blood clotting factor VIII
human growth hormone
bovin chymosin
hepatitis B vaccine
Artemisinin

Question 22

What is synthetic biology?

Answer 22

design and construction of new biological parts, devices and systems, and the redesign of existing natural biological systems for useful purposes

using synthetic biology and genetic engineering can answer biological questions and produce essential gene products in the lab

Question 23

Artemisinin production

Answer 23

creating of anti-malerial drug
from artemisia annua- sweet wormwood used by Chinese herbal practitioners for years