DNA Profiling Flashcards

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1
Q

What is a genome?

A

-all of the genetic material that it contains

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2
Q

What are genes?

A

-regions of DNA on chromosomes that code for polypeptides

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3
Q

What are introns?

A

-larger, non coding regions of DNA in between the genes

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4
Q

What is a tandem repeat?

A

Repetitive sequences of DNA that do not code for proteins found within introns

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5
Q

What is a variable number tandem repeat (VNTR)

A

-A location in a genome where a short nucleotide sequence is organised as a tandem repeat
-these can be found on many chromosomes, and often show variations in length (number of repeats) among individuals
-they are 10-100 base pairs long, repeated 50 to several hundred times

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6
Q

What are short tandem repeats?

A

-smaller repeated sequences within the non-coding DNA
-they are 2-9 base pairs long and are repeated 5 to 15 times

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7
Q

Why does the number of short tandem repeats vary between individuals?

A

-because we inherit different lengths of repeats from each parent
-it is these different patterns that is used in DNA profiling

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8
Q

Explain the first step of the method for DNA profiling

A

1) Extract the DNA
-from saliva, blood drop, hair, semen, bone sample (only tiny amounts are needed because lab techniques can copy the DNA many times to give a large sample to analyse)
-then the DNA needs to be purified because the histone proteins need to be removed using protease enzymes before the DNA is sequenced

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9
Q

Explain the second step in the method for DNA profiling

A

2) Digest the sample with enzymes
-the DNA sample is cut into smaller fragments using restriction enzymes
-they are always cut at specific base pairings because the shape of the enzymes active site will be complementary to the shape of a particular sequence of bases in the DNA, so an enzyme substrate complex can form and the relevant bonds are broken

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10
Q

Explain why restriction enzymes come from bacteria?

A

Bacteria have them as it stops viral DNA taking over the cell as it can be broken down into little pieces helping the cell survive

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11
Q

Explain the third step in the method for DNA profiling.

A

3) Separation of the DNA fragments: Gel electrophoresis
-the fragments need to be separated in order to look for patterns that can then be analysed, done through a technique called Electrophoresis

-DNA fragments are put into a ‘well’ in a block of gel, along with some loading dye to make the DNA visible
-this is placed in an alkaline buffer solution, which regulates pH and helps to carry the electrical charge across the gel
-the gel then has an electric current pass through it
-the DNA fragments move towards to positive end (because phosphate groups in DNA give a negative charge)
-the smaller the fragment, the further it moves as has less resistance to gel
-it is important to let it run for sufficient time to allow all the DNA fragments to separate out fully

The DNA is then made single stranded ready for analysis
-a nylon membrane is then placed on top of the gel, with absorbent paper on top. This draws the top strands of DNA onto the nylon = Southern blotting
-the DNA strands are fixed in place onto the nylon membrane by either UV light or heat at 85 degrees

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12
Q

Explain the fourth step in the method for DNA profiling

A

4) Hybridisation
-now DNA probes are added to look for particular mini/micro satellites in the DNA
-the probe has a complementary base sequence to the sequence you are looking for
-when they attach = hybridisation
-the probes are usually used to locate the microsatellites in the DNA because these are more variable from person to person
-any excess probes are washed away

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13
Q

What is a probe?

A

-stretches of single stranded DNA used to detect the presence of complementary nucleic acid sequences by hybridisation
-they are usually labelled with radioisotopes or fluorophores to enable their detection
-they are 100- 1000 base pairs long

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14
Q

Explain the fifth step in the method for DNA profiling

A

5) making it visible
-we cannot see DNA, but if the probes used are radioactive or fluorescent they can be detected wherever they have attached
-if radioactive probes are used placing the nylon membrane onto an X-ray film causes the film to fog where the radiation is
-if fluorescent probes were used, place the nylon membrane under UV light and the probes will glow
-usually at least 13 different STR’s will be looked for when doing a DNA profile. This ensures the wrong person isn’t found as the chances of having 12 different STR’s is hundreds of millions to one

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15
Q

What is the PCR (Polymerase chain reaction) used for?

A

Making multiple copies of the DNA sample so there is enough to analyse

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16
Q

What is needed for the PCR technique?

A

-original tiny DNA sample
-excess of free nucleotides with the 4 different bases
-primers (short pieces of single-stranded DNA which start the copying process) 12-15 nucleotides long
-DNA polymerase enzyme (Taq polymerase as comes from thermophilic hot-spring bacteria so not denatured by high temperatures in the machine)

-all of these are mixed together in a small tube and placed into the PCR machine

17
Q

What is the first step for PCR technique? Include temperature needed

A

-temperature inside is 90-95 degrees for 30 seconds
-this breaks the hydrogen bonds between the 2 strands of DNA, separating them

18
Q

What is the second step for PCR technique? Include temperature needed

A

-temperature decreased to 55-60 degrees
-primers join to both ends of the single stranded DNA (short single stranded section of DNA complementary to the ends of the strands needed for replication to start)

19
Q

What is the third step for PCR technique? Include temperature needed

A

-temperature increased to 70-75 degrees for 1 minute
-this is the optimum temperature for the chosen DNA polymerase enzyme (Taq polymerase) to work
-this adds bases to the primers, extending the complementary strands
-this results In double stranded DNA genetically identical to the original sample

20
Q

Why might you not get as many copies of the DNA made as expected?

A

-may not be enough primers present
-primers might not attach to all of the DNA strands
-may be insufficient nucleotides available
-2 separated DNA strands may just rejoin to each other rather than the primers

21
Q

What is a similarity and difference between probes and primers?

A

-similarity = both single stranded sections of DNA
-difference =
probes are used to locate a particular base sequence + 100 to 1000 nucleotides long
Primers are used to start DNA replication of the DNA strands in PCR and are 15 to 25 nucleotides long