Genetic Tools Flashcards

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1
Q

What is PCR?

A

a test for the amplification of DNA

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2
Q

what are the 3 steps in PCR?

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

repetition of pcr

A

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

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4
Q

Amplification

A

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

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5
Q

What are the PCR primers?

A

short
20bp
single stranded DNA
chemically synthesised

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6
Q

what is agarose gel electrophoresis?

A

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

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7
Q

separation of DNA by size

A

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

stain DNA with fluorescent dye for detection by UV exposure

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8
Q

What is Sanger Sequencing?

A

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

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9
Q

What are the uses of PCR?

A

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

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10
Q

What are the limitations of PCR?

A

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

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11
Q

Key points of PCR

A

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

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12
Q

what are plasmids?

A

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

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13
Q

How do you cut DNA?

A

by restriction endonucelases
creating 5’ overhang

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14
Q

How do you join DNA?

A

DNA ligase
forming phosphodiester bonds
requiring ATP

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15
Q

What is recombinant DNA?

A

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

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16
Q

What is gene cloning?

A

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

17
Q

Collection of recombinant clones

A

canoe frozen and stored
creating a gene library for future use

18
Q

What is transgenics?

A

genes between species
the genetic code is universal between organisms

19
Q

How can DNA be expressed in cells?

A

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
20
Q

How do you make CDNA?

A
  1. Extract RNA
  2. Reverse transcribe
  3. Treat with RNAse H
  4. Make second strand of DNA with DNA polymerase
21
Q

What are examples of genetic engineering?

A

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

22
Q

What is synthetic biology?

A

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

23
Q

Artemisinin production

A

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