Unit 7- Analysing DNA Flashcards

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1
Q

What is the purpose of PCR?

A

To make large number of copies of DNA fragments

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2
Q

In PCR why do we use primers? And why is the primer we chose important

A
  • primers act as a binding point for DNA polymerase, as it can only bind to double stranded DNA
  • only the DNA between primers is replicated, so we need to chose a primer than doesn’t overlap the gene that we are trying to replicate
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3
Q

Why isn’t DNA polymerase denatured at the high temps of PCR? And what’s this enzymes optimum temp?

A
  • the DNA polymerase used derives from a thermophillic bacterium that lived in hot springs (90 degree)
  • optimum temp is 72 degrees c
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4
Q

Describe the process of PCR

A
  • solution of DNA fragments is heated to 90-95 degrees C for 30s
  • cooled to 50-55 degrees C, primers bind
  • heat to 72 degrees C for DNA polymerase to build complementary strand
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5
Q

In PCR why do we heat to 95 degrees, then add primers and cool to 50 degrees, and then reheat to 72 degrees?

A

95 degrees breaks hydrogen bonds
At 50 degrees hydrogen bonds can form between strand and primers
72 degrees is the optimum temp for DNA polymerase

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6
Q

In PCR after 3 repeats how many times more strands of DNA are there?

A

2^3

So 8, as its 2^n, when n is the number of times the process is repeated

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7
Q

Positive and negatives of PCR

A

+ can be used to make more from a tiny sample, useful in forensics
- the sample needs to be pure or contaminant DNA will also be amplified

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8
Q

What is the difference between PCR and Gene sequencing

A

Gene sequencing uses terminator bases with fluorescent tags and the sequence is worked out with computers
PCR replicates the sequence

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9
Q

Describe the process of gene sequencing

A
  • samples of fragmented DNA are heated to 95 degrees C to break hydrogen bonds
  • sample cooled to 50 so primers can anneal
  • sample reheated to 72 degrees do DNA polymerase builds the strands until a terminator base stops the sequence
  • sequence can be determined using by separating out fragments via gel electrophoresis or computers
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10
Q

What is a terminator base?

A

A nucleotide with an oxygen missing so the phosphodiester bond cannot form on one side so the sequence can’t continue

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11
Q

Why do we use DNA sequencing?

A
  • to compare sequences between species to see how closely related
  • to compare sequences between individuals to deduce family relation
  • to identify allleles linked with genetic diseases
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12
Q

What is an issue with gene sequencing?

A

Predicting amino acid sequences is hard because introns are removed from DNA in splicing

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13
Q

What is DNA profiling used for?

A
  • used in forensics and paternity tests
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14
Q

Explain what STRs and VNTRs are

A

Regions of non coding DNA that contain simple repetitive sequences

  • short Tandem repeats (STRs) are when a sequence of about 4 nucleotides are repeated about 3-15 times
  • everyone has STR sequences in the same loci but different people have different numbers or repeats
  • STR regions are called (VNTR), variable number tandem repeats
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15
Q

Describe the process of DNA profiling

A

1- begin with the sample of DNA
2- amplify the sample of DNA via PCR using special primers that bind at the start of the VNTR sequence so the repetitive sequence is replicated
3- use restriction endonucleases to cut the DNA into unique fragments
4- seperate the fragments by electrophoresis
5- southern blot the agar
6- light emitted by fluorescent primers are detected by sensor

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16
Q

What can DNA profiling be used for?

A
  • in forensics to match DNA samples to criminals
  • paternity tests
  • to prevent inbreeding in zoos
  • to measure genetic diversity
  • to establish phylogenetic relationships between species
17
Q

What is electrophoresis?

A

Separating different pieces of DNA on the basis of their length using electric currents

18
Q

Describe what happens in electrophoresis

A
  • DNA samples are placed into wells at one end of the slab of the agar
  • covered in a buffer solution
  • an electric current is passed through the gel
  • DNA is negatively charged due to the phosphate group so diffuses through the gel to the positive electrode
  • the longer the strand of DNA the slower it diffuses
19
Q

How can we make it so we can see the bands produced by electrophoresis??

A
  • stain the DNA with azure which is blue

- stain the DNA with a fluorescent molecule (ethidium bromide) which emits colour under UV

20
Q

What is the purpose of the southern blot?

A

To detect specific target sequences in a sample of DNA

21
Q

What is a DNA probe?

A

A single Strand of DNA with radioactive/fluorescent label that will anneal to its complementary sequence

22
Q

What is hybridisation?

A

When a DNA probe anneals to its complementary strand forming double stranded hybrid DNA

23
Q

Describe the southern blot method

A

1- DNA is extracted and amplified by PCR
2- the dna is digested by restriction endonucleases to make smaller fragments
3- fragments are separated by gel electrophoresis after adding alkali to break the double stranded fragments into single stranded DNA
4- a sheet of nylon is placed on the gel, the DNA sticks to the nylon membrane
5- the nylon is placed in a solution of the labelled probes which will anneal to their complementary strands on the nylon forming hybrid DNA
6- the location of the hybrid DNA can be seen using a UV light as the labels may be fluorescent or auto radiography if radioactive labels were used