Section 8 - Recombinant DNA technology Flashcards
What is gene sequencing?
Identifying the order of the base pairs in a segment of DNA
What is the Sanger method?
A technique which uses modified nucleotides that cannot attach to the next base sequence when they are joined together
They act as terminators and stop synthesis of a DNA strand
What does DNA sequencing involve?
Cutting DNA with restriction enzymes
Separating fragments by electrolysis
Coping by polymerase chain reaction
What is the basic method of sequencing genes?
4 test tubes are labelled T, G, A, C, each has DNA polymerase, primer, modified nucleotide terminator, normal nucleotides and lots of single stranded DNA
Gel electrophoresis separates DNA fragments and identified due to labelled primer
Larger fragments move more slowly and travel least distance
What is within each test tube in the Sanger method and what is the function of each?
DNA polymerase - joins nucleotides of new strand together
Primer - keeps DNA strands separate and enables sequencing to start
Modified nucleotide terminator
Normal nucleotides
Lots of single stranded DNA fragments to be sequenced
What is genome sequencing?
Identifying DNA base sequence of an individual
Allows us to determine the amino acid sequence of the polypeptides coded for by that DNA
How are DNA fragments separated using electrophoresis?
DNA samples are treated with restriction enzymes to cut them into fragments and pipetted into a ‘well’
DNA is negatively charged due to phosphate groups so it is attracted to the positive electrode
Shorter DNA lengths move faster so move further
How is the position of fragments shown in electrophoresis?
Shown using a dye that stains the DNA molecules
What is southern blotting?
Nylon or nitrocellulose sheet is placed over agar gel
DNA fragments are transferred to the sheet and can be analysed with radioactive marker
Placing photographic film over nitrocellulose sheet shows position of DNA samples in finished gel
What is the resulting cycle sequencing solution separated with?
Using capillary electrophoresis
How is cycle sequencing gel read?
By a laser beam
Sequence of colours is converted to a DNA sequence by computer program
What is the reaction used to amplify small amounts of DNA into quantities large enough for Sanger procedure?
Polymerase chain reaction
When a modified nucleotide is used to form a complementary DNA strand, the sequencing reaction is terminated.
Suggest how this sequencing reaction is terminated?
Missing nitrocellulose
So no phosphodiester bond
What makes DNA fragments visible on an autoradiogrpah?
Primer is radioactive
By what processes can fragments of DNA be produced?
Using reverse transcriptase
Using restriction enzymes
Gene machine
What does the recombinant of DNA technology involve?
Involves the transfer of fragments of DNA from one organism to another
How can reverse transcriptase be used to make fragments of DNA?
Isolate cell that naturally produces that protein to collect the mRNA from the sample
Complementary DNA (cDNA) is made from the mRNA that is a template
cDNA is combined with DNA polymerase which will makes the complimentary strand of DNA
What enzyme do retroviruses like HIV contain?
Reverse transcriptase
How can restriction enzymes be used to makes fragments of DNA?
Enzymes cut DNA at specific sites (restriction sites)
Restriction site is normally a palindromic sequence
Restriction enzymes recognise a specific sequence and cut the sugar phosphate backbone
Exposed base = sticky ends - enable 2 strands of DNA to be easily joined together by complimentary base pairing
Some restriction enzymes result in blunt ends that do not have exposed bases
What do restriction enzymes do in bacteria?
Protect themselves from phage viruses
What can restriction enzymes be used for?
To isolate a specific gene by cutting it form the larger DNA molecule
What are sticky ends and what do they enable?
Exposed bases
Enable 2 strands of DNA to be easily joined together by complimentary base pairing
What does a gene machine do?
Chemically synthesizes single-stranded DNA (ssDNA)
Synthetic double-stranded DNA (dsDNA) can then be made
Creates DNA strands with no introns
What is the maximum length of ssDNA?
100 nucleotides
What are oligonucleotides?
Overlapping sequences
What does the gene machine remove?
Removes the need to convert mRNA to DNA or use restriction enzymes
What are the other uses of the gene machine?
Sequence can be synthesised and inserted into DNA sequence to add a restriction site where it otherwise would not occur
Synthesising altered sequence of bases that results from a gene mutation to study the effects on the polypeptide
Synthesising DNA probs to be used in genetic screening
How might the production of synthetic genes be useful?
Gene therapy
Drugs produced
Allows current genes to be refined and made more efficient
What is the enzyme used to join ends of DNA together?
DNA ligase
In genetic engineering, why is it preferable to use DNA with sticky ends when this reaction is carried out?
Allows complementary bindings which gives specificity
What is a practical use of PCR?
Crime scene investigation
What are 2 ways in which PCR differs from the process of transcription?
Doesn’t involve helicase
Don’t get mRNA
What 4 things are used in the PCR process?
Taq polymerase - used as stable at high temperatures
Primer - single stranded DNA which is complementary to a specific sequence at the 3’ end of the DNA that is to be replicated
Nucleotides
DNA
What is the process of PCR?
- DNA strands separated by heating to 95’c for 2 mins (breaks hydrogen bonds)
- DNA cooled to 55’c to cause primers to anneal (base pair) to DNA
- Temperature increased to 72’c which is optimum temperature for the polymerase
- DNA polymerase extends the primers and replicated remaining DNA
- Each original DNA molecule is replicated so with each cycle, number of DNA molecules doubles
How many copies of DNA would be made after:
a. 5 cycles
b. 10 cycles
a. 32
b. 1024
Describe how altered DNA may lead to cancer? [6]
DNA altered by mutation causes a change in the base sequence of genes that monitor cell division
Change in protein structure, tumour suppressor genes produce proteins that inhibit cell division
Uncontrolled rapid cell division forming malignant tumor
Explain why fragments of DNA from cancer cells may be present in blood plasma?
Cancer cells die releasing DNA
Explain how examining mRNA enables scientists to discover whether cancer is present? [3]
mRNA base sequence is changed
DNA structure is different
Tumour suppressor gene is inactive
