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  • Created by: Amira200
  • Created on: 02-06-16 10:05
Which enzymes are required to cut DNA strands into smaller fragments
Restriction endonuclease enzymes
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What is the name of the process for separating DNA fragments by size
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What is the process called for replicating fragments to make multiple copies
Polymerase chain reaction
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Which enzymes are required to seal DNA fragments together
DNA ligase enzymes
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What is the use of DNA probes
Locate specific sequences on DNA fragments
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Why is the term junk DNA misleading
Because although this DNA is non coding DNA it still takes part in carrying out a number of regulatory functions
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Why do genomes need to be broken up and sequenced into sections
Because sequencing process only works on a length of DNA up to 750 base pairs long
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What are micro satellites
Short runs of repetitive sequences of 3-4 base pairs found in several thousand locations on the genome
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Why is sequencing carried out many times on overlapping fragments
To ensure accuracy
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Why are different restriction enzymes used
So that different fragment types are produced. The fragments will then overlap allowing the overlapping regions to be analysed and resequenced to form a completed code
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What is comparative gene mapping
When you know the sequence of bases in a gene of one organism and be able to compare genes for the same or similar proteins across a range of organisms.
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How does gene sequencing allow for genome wide comparisons between individuals and species
Identification of genes of proteins give relative importance of such genes to life.Shows evolutionary relationships.Modelling the effects of changes to DNA.Identify the genes that are most important in causing disease.Reveal mutant alleles
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Describe electrophoresis
Separating DNA fragments into size order. Fragments are negatively charged so move towards the anode (positively charged side). Smaller fragments move the fastest and the furthest
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Name three vital things in an electrophoresis tank and explain why essential
Agarose gel which allows fragments to move down. Buffer solution which controls temperature changes. Electrodes so that a current can be passed through it.
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Why is DNA negatively charged
DNA has many phosphate groups which are negatively charged
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Which fragments move fastest and why
The smallest fragments move the fastest and furthest through the gel because they don't get caught up in the gel like larger fragments which as a consequence move slower and less further
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How can the position of fragments be shown
Using a dye that stains the DNA molecules
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What is the technique called for when fragments are lifted from gel for further analysis
Southern blotting technique. A nylon sheet placed over gel and left over night to blot. Then radioactive markers which have been labelled on the DNA will identify the DNA fragments.
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What is a DNA probe
A short single stranded piece of DNA that is around 50-80 nucleotides long and complementary to a piece of DNA being investigated.
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How is a DNA probe labelled
Using a radioactive marker (location revealed by exposure to photographic film) or a fluorescent marker(emits colour on exposure to UV light)
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Because probes are single stranded what will they do
They will bing by complementary base pairing to any fragment where complementary base sequence is present. Binding is known as annealing
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What is binding by complementary base pairing also known as
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Why are probes useful
They can locate a specific desired gene that is wanted for genetic engineering. Genome wide comparisons. Identify presence of absence of an allele for particular disease.
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What is DNA microarray
A fixed surface which scientists place a number of different probes on to reveal the presence of faulty or mutated alleles that match the fixed probes
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Where is PCR particularly useful
In crime scene investigations where samples of DNA are taken and multiplied to generate enough material for genetic profiling
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The sequencing reaction in PCR relies on the many features of DNA What are these features
Made up of antiparallel backbone strands. Made of strands with 5" end and 3" end. Grows from only 3" end. Base pairs pair up by CBP
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What are the weaknesses of PCR
It can only replicate short sequences of DNA not an entire chromosome. Primers are required in order to start process. A cycle of heating and cooling is used to separate and ind strands.
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In natural DNA replication what is used to separate strands
DNA helicase enzyme
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What are primers
Short single stranded sequences of DNA around 10-20 bases in length
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Why are primers required in PCR
To bind to a section of DNA because the DNA polymerase enzymes cannot bind directly to single stranded DNA fragments
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Why is DNA polymerase described as thermophilic in PCR
It doesn't denature at high temperatures used in the process.
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What happens if a modified nucleotide is added during PCR
Polymerase enzyme is thrown off and the reaction stops on that template strand
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Define the term recombinant DNA
A section of DNA formed by combining DNA sections from 2 different organisms
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Describe restriction enzymes
Endonuclease enzyme used to cut DNA and expose sticky ends at a specific restriction site from bacteria
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What are exposed bases called and how are they formed
Sticky ends. Restriction enzymes catalyses a hydrolysis reaction which breaks phosphate sugar backbone of DNA double helix in different places leaving a staggered cut
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What is the name of the enzyme that stick DNA fragments together
DNA ligase
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Describe DNA ligase
Catalyses a condensation reaction which joins phosphate sugar backbone of DNA double helix together
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What do the DNA fragments have to have in order to be joined back together
Need to be cut by the same restriction enzyme so there sticky ends are complementary and allows bases to pair up and hydrogen bond togethor. DNA ligase can then seal the backbone
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Describe the role of DNA polymerase
Makes single traded DNA double stranded. DNSA replication by PCR. using DNA polymerase with high optimum temp
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Describe the role of DNA ligase for 2 marks
Seals nicks in DNA backbone and joins sugar to phosphate
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What is reverse transcriptase
Forms DNA from mRNA
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Describe the role of restriction enzymes for 4 marks
Cut gene from genome and cut plasmid. Use same restriction enzyme. If cut with sticky ends then sticky ends join. If cut with blunt ends then sticky ends added. with c bases one end and g bases the other. Requires terminal transferase
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How is recombinant DNA formed
2 DNA sources. sticky ends. complementary binding of bases. H bonds form betweenness. A-T, C-G. Sugar phosphate backbone resealed by DNA ligase
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What does genetic engineering involve
The extraction of genes from one organism and placing them into another organism
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How are sections of DNA containing a desired gene extracted from donor organism
Restriction enzymes
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Briefly explain the steps involved in genetic engineering
1) Obtain gene 2)Place gene in vector 3) Place vector containing gene into recipient cell 4)Identify transformed bacteria that the recipient has expressed through protein synthesis
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Describe step 1 (obtaining gene) of genetic engineering
Use DNA probe to locate gene. Isolate desired DNA fragment containing gene using restriction enzymesD
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Describe step 2 (Place gene into vector)
Insert DNA fragment into vector DNA. Use same restriction enzyme to open up veto DNA. DNA ligase seals vector DNA and DNA fragment by sugar phosphate backbone. Recombinant DNA formed
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Describe step 3 (Place gene into recipient cells)
Recipient cells added to CaCl2 solution to make cell walls more permeable. Plasmids added and heat shocked to encourage cell to take up plasmids
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Describe step 4 (Identify transformed bacteria)
Recipient expresses gee through protein synthesis meaning that the transformed bacteria can be recognised using marker genes
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Gene packaged in vector can form a large molecule that can't easily cross membrane to enter recipient cell. What are methods to overcome this
Microinjection. Electroporation. Viral transfer. Liposome. DNA is wrapped round lipid molecules which are fat soluble and cross lipid membrane by diffusion
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Name some other vectors
Bacterial plasmids. Yeast cell chromosomes. Virus genomes
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Why do we genetically engineer
Improve feature of recipient organism . Engineering organisms can produce useful products e.g. insulin
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