Recombinant DNA technology Flashcards

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1
Q

How can plasmids be genetically modified and inserted into bacteria

A
  • isolate the wanted gene from the DNA
  • using restriction endonuclease to leave complementary sticky ends on the selected gene and the plasmid
  • insert the gene into plasmid using ligase enzyme with the addition of a marker e.g. antibiotic resistance
  • put the plasmid containing the recombinant DNA back into the bacteria by heating then cooling then heating
  • grow on a medium where marker would be expressed e.g. containing antibiotics
  • bacteria that grows will have be antibiotic resistant and contain the desired gene
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2
Q

Name the type of enzyme used to cut open the plasmid

A

restriction endonuclease

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3
Q

Name the type of enzyme used to insert the gene onto the plasmid

A

ligase

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4
Q

Describe the process of genetic fingerprinting

A
  • DNA is cut into fragments using a restriction enzyme
  • DNA fragments are separated according to size by gel electrophoresis under electrical influence
  • gel is then immersed into alkali to separate the double strands into single strands (southern blotting) and then transferred onto a nylon membrane
  • radioactive/fluorescent DNA probs are used to bind to the complementary base sequence of the VNTRs under specific temperature and pH conditions
  • nylon film can be x-rayed and the probs will be exposed, fluorescent probe can be located visually
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5
Q

What is the process of PCR (Polymerase Chain Reaction)

A

increases the amount of DMA in a sample by:

  • DNA strand is separated and the DNA fragments, primers and DNA polymerase are placed in a vessel, and heated to 95°C
  • the mixture is cooled to 55°C, causing the primers to anneal (join)to their complementary bases at the ends of the DNA fragment
  • the synthesis of DNA happens when the temperature is increased to 72°C which is the optimal temperature for the DNA polymerase to add complementary nucleotides to the DNA strands
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6
Q

What is a primer of the PCR

A
  • short sequences of nucleotides that have a set of bases complementary to those at one end of the two DNA fragments
  • provide a starting point for the DNA polymerase to begin copying the DNA
  • prevent two separate DNA strands from rejoining
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7
Q

What is in vitro cloning

A

using method of PCR that is rapid and does not require living cells

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8
Q

What is in vivo cloning

A

isolating the gene of interest and use of gene machine

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9
Q

What is meant by the term Variable Number Tandem Repeat (VNTR)

A

highly repetitive sequences of DNA bases

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10
Q

How does the structure of a genome allow it to be used for genetic fingerprinting

A
  • VNTRs differ in size/length between different individuals
  • VNTRs occur in lots of different places in the genome between different individuals
  • the difference in size and location of VNTRs creates a genetic fingerprint unique to the individual which can be used to identify them
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11
Q

What is southern blotting used for

A

to separate double-stranded DNA, to make it single-stranded for the DNA probe to bind

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12
Q

How is a gene made using a gene machine

A
  • the amino acid sequence os determined
  • the nucleotide triplets are worked out and the base sequence is fed into a computer
  • the computer designs gly
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13
Q
Describe the following stem cells:
Totipotent
Pluripotent
Multipotent
Unipotent
A

totipotent - can differentiate into any type of cell, found in early embryo

pluripotent - can differentiate into almost any type of cell, found in embryonic and fetal stem cells

multipotent - can differentiate into limited number of specialised cells, found in adult and umbilical cord blood stem cells

unipotent - can differentiate into a single type of cell, found in adult tissue

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14
Q

What are induced pluripotent stem cells (iPS cells)

A
  • they are very similar to embryonic stem cells (pluripotent cells) however they can potentially divide indefinitely
  • can overcome ethical issues surrounding use of embryonic stem cells
  • can be used to regrow tissues
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15
Q

What is decreased acetylation

A
  • acetyl group is removed from molecule
  • increases positive charges on histones and therefore increases attraction to the phosphate groups of DNA
  • DNA becomes less accessible to transcription factors to initiate mRNA productions
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16
Q

What is increased methylation

A
  • methyl group is added to molecule
  • inhibits transcription by making DNA inaccessible to transcription factors
  • by attracting proteins that condense the DNA-histone complexes
17
Q

What are causes of uncontrollable cell devision

A

proto-onco genes that mutate are called oncogenes:

  • this oncogene may code for a receptor that does not need a growth factor
  • or oncogene may code for to much growth factor

a mutated tumour suppressor gene may cause cell devision not to be switched off thus not coding for an inhibitor of a growth factor

18
Q

Why are they a variety of different primers used in an experiment

A

as the base sequence of nucleotides differ, different complementary primers are needed

19
Q

Why would DNA replication in the PCR stop

A

as the primers/nucleotides run out