Gene technology

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Recombinant DNA
A combination of DNA from two different organisms.
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Transgenic/GM/Transformed
An organism that contains DNA from two different organisms.
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5 Steps in DNA transfer
1 - Isolation, 2 - Insertion, 3 - Transformation, 4 - Identification, 5 - Growth/cloning.
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What is unique about restriction enzymes?
They have highly specific active sites, which are complementary to a certain recognition site on a DNA strand.
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Palindromic
Where a base sequence and its complement are the same but reversed.
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Examples of restriction endonucleases
Eco - RI cuts at GAATTC. Hind III cuts at AAGCTT.
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3 Methods of isolation
1) Using reverse transcriptase. 2) Using restriction enzymes. 3) Using a gene machine
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Vector
A carrier - e.g. a plasmid that carries DNA into a cell.
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Which enzyme re - forms bonds between DNA sugar - phosphate backbone molecules when the required gene and plasmid are spliced together?
DNA ligase.
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What is produced when reverse transcriptase acts on mRNA?
cDNA (complementary DNA)
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What is mixed together in the first step of transformation?
Bacteria (to become GM), plasmids and ice cold calcium chloride solution.
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Why is the solution used in transformation heat - shocked?
So that the bacteria become permeable and the plasmid can pass across their membranes into the cell.
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Why might bacterial cells not take up the desired recombinant DNA?
Some plasmids close up before incorporating the DNA fragment, and sometimes DNA fragment ends join together to form its own plasmid.
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3 types of gene marker
A fluorescent protein (GFP), an enzyme whose action can be identified, a gene giving resistance to a certain antibiotic.
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Why are the same restriction endonucleases used to cut the plasmid and the required gene?
So that the plasmid and required gene have complementary sticky ends.
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Which enzyme is used as a gene marker?
Lactase
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Which colourless substrate does lactase act on?
Turns X - gal blue (chemical name BCIG)
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Which bacteria fluoresce when the GFP gene has been added to their plasmids? *
The bacteria that do not contain the desired DNA fragment (as the fragment has not been spliced into the middle of the GFP gene).
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Requirements for PCR
DNA fragment to be copied, DNA polymerase, primers, DNA nucleotides, thermocycler
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Where is DNA polymerase obtained to be used in PCR?
Bacteria that live in hot springs - so the DNA polymerase is tolerant to heat.
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What are primers?
Short pieces of DNA that bind to the start of each end of fragment and signal to a polymerase where to start synthesising new DNA. They also help to prevent both ends from joining back up.
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What is a thermocycler?
A computer controlled machine that varies temperature precisely for set time periods.
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3 temperatures used in PCR
95 degrees - for separation of DNA strand. 55 degrees - for addition of primers. 72 degrees - for synthesis of DNA.
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What are the blocks of repeated nucleotides contained in introns called?
Variable Number Tandem Repeats (VNTRs).
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Stages of gel electrophoresis (Every Day Should Have Development)
Extraction>digestion>separation>hybridisation>development.
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Which element is used in a radioactive DNA probe?
P32
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Which base sequence gets repeated in Huntington's disease? (Alan Goes Camping)
AGC
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How many repeats of the 3 base sequence must someone have to suffer from Huntington's?
< 30 repeats = unlikely to have disease. >/38 repeats = likely to have disease. >/50 repeats = likely to suffer from early - onset Huntington's.
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How can painkillers be personalised?
An enzyme is needed to activate certain painkillers. Patients can be screened for the gene encoding this enzyme to see which painkiller and which dosage will be effective.
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How can vitamin E be used in personalised medicine?
Diabetics with certain genotypes will have a reduced risk of cardiovascular disease with vitamin E supplements. However, this is opposite with some other genotypes.
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2 types of gene therapy
Somatic cell therapy and germ line cell therapy.
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What mutation causes cystic fibrosis? (Alan's Always Angry)
A deletion mutation of 3 bases (AAA) in the CFTR gene.
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What is the role of the CFTR protein?
To transport Cl- ions across epithelial cell membranes.
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2 types of vector that can be used in gene therapy
Adenovirus (harmless virus) or liposomes.
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What is done before hybridisation? (stage 4 of gel electrophoresis)
The gel is immersed in alkali in order to separate the the double strands into single strands (exposing the DNA bases so that complementary probes can attach).
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Why are primers needed for synthesis of new DNA?
To create a double - stranded section for the DNA polymerase to begin synthesis from - polymerase cannot act on single - stranded DNA.
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What must be added to fragments of DNA before insertion into vectors and why?
Promoter and terminator regions - to ensure that only the required gene from the DNA fragment is expressed in the recipient organism.
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Other cards in this set

Card 2

Front

Transgenic/GM/Transformed

Back

An organism that contains DNA from two different organisms.

Card 3

Front

5 Steps in DNA transfer

Back

Preview of the front of card 3

Card 4

Front

What is unique about restriction enzymes?

Back

Preview of the front of card 4

Card 5

Front

Palindromic

Back

Preview of the front of card 5
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