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Biology > Section 8 - Recombinant DNA technology > Flashcards

Section 8 - Recombinant DNA technology Flashcards

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1
Q

What is gene sequencing?

A

Identifying the order of the base pairs in a segment of DNA

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2
Q

What is the Sanger method?

A

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

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3
Q

What does DNA sequencing involve?

A

Cutting DNA with restriction enzymes

Separating fragments by electrolysis

Coping by polymerase chain reaction

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4
Q

What is the basic method of sequencing genes?

A

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

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5
Q

What is within each test tube in the Sanger method and what is the function of each?

A

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

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6
Q

What is genome sequencing?

A

Identifying DNA base sequence of an individual

Allows us to determine the amino acid sequence of the polypeptides coded for by that DNA

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7
Q

How are DNA fragments separated using electrophoresis?

A

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

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8
Q

How is the position of fragments shown in electrophoresis?

A

Shown using a dye that stains the DNA molecules

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9
Q

What is southern blotting?

A

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

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10
Q

What is the resulting cycle sequencing solution separated with?

A

Using capillary electrophoresis

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11
Q

How is cycle sequencing gel read?

A

By a laser beam

Sequence of colours is converted to a DNA sequence by computer program

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12
Q

What is the reaction used to amplify small amounts of DNA into quantities large enough for Sanger procedure?

A

Polymerase chain reaction

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13
Q

When a modified nucleotide is used to form a complementary DNA strand, the sequencing reaction is terminated.

Suggest how this sequencing reaction is terminated?

A

Missing nitrocellulose

So no phosphodiester bond

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13
Q

What makes DNA fragments visible on an autoradiogrpah?

A

Primer is radioactive

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14
Q

By what processes can fragments of DNA be produced?

A

Using reverse transcriptase

Using restriction enzymes

Gene machine

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15
Q

What does the recombinant of DNA technology involve?

A

Involves the transfer of fragments of DNA from one organism to another

16
Q

How can reverse transcriptase be used to make fragments of DNA?

A

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

17
Q

What enzyme do retroviruses like HIV contain?

A

Reverse transcriptase

18
Q

How can restriction enzymes be used to makes fragments of DNA?

A

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

19
Q

What do restriction enzymes do in bacteria?

A

Protect themselves from phage viruses

20
Q

What can restriction enzymes be used for?

A

To isolate a specific gene by cutting it form the larger DNA molecule

21
Q

What are sticky ends and what do they enable?

A

Exposed bases

Enable 2 strands of DNA to be easily joined together by complimentary base pairing

22
Q

What does a gene machine do?

A

Chemically synthesizes single-stranded DNA (ssDNA)

Synthetic double-stranded DNA (dsDNA) can then be made

Creates DNA strands with no introns

23
Q

What is the maximum length of ssDNA?

A

100 nucleotides

24
Q

What are oligonucleotides?

A

Overlapping sequences

25
Q

What does the gene machine remove?

A

Removes the need to convert mRNA to DNA or use restriction enzymes

26
Q

What are the other uses of the gene machine?

A

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

27
Q

How might the production of synthetic genes be useful?

A

Gene therapy

Drugs produced

Allows current genes to be refined and made more efficient

28
Q

What is the enzyme used to join ends of DNA together?

A

DNA ligase

29
Q

In genetic engineering, why is it preferable to use DNA with sticky ends when this reaction is carried out?

A

Allows complementary bindings which gives specificity

30
Q

What is a practical use of PCR?

A

Crime scene investigation

31
Q

What are 2 ways in which PCR differs from the process of transcription?

A

Doesn’t involve helicase

Don’t get mRNA

32
Q

What 4 things are used in the PCR process?

A

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

33
Q

What is the process of PCR?

A
  1. DNA strands separated by heating to 95’c for 2 mins (breaks hydrogen bonds)
  2. DNA cooled to 55’c to cause primers to anneal (base pair) to DNA
  3. Temperature increased to 72’c which is optimum temperature for the polymerase
  4. DNA polymerase extends the primers and replicated remaining DNA
  5. Each original DNA molecule is replicated so with each cycle, number of DNA molecules doubles
34
Q

How many copies of DNA would be made after:

a. 5 cycles
b. 10 cycles

A

a. 32

b. 1024

35
Q

Describe how altered DNA may lead to cancer? [6]

A

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

36
Q

Explain why fragments of DNA from cancer cells may be present in blood plasma?

A

Cancer cells die releasing DNA

37
Q

Explain how examining mRNA enables scientists to discover whether cancer is present? [3]

A

mRNA base sequence is changed

DNA structure is different

Tumour suppressor gene is inactive

Biology (25 decks)

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