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biology > Genetic techniques- Module 6 > Flashcards

Genetic techniques- Module 6 Flashcards

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AP® Biology flashcards
1
Q

Primers-

A

short pieces of DNA complementary to the bases at the start of the fragment of DNA needed

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2
Q

Taq polymerase-

A

enzyme that creates new DNA strands. It is thermostable so it doesn’t denature at high temperatures.

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3
Q

stAGE 1 OF pcr (DENATURING)

A

DNA mixture is heated to 95oC to break the hydrogen bonds between the two strands of DNA (they uncoil).

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4
Q

stAGE 2 OF pcr (Annealing)

A

The mixture is then cooled to 50-65oC so that the primers can bind (anneal) to the strands. to recognise target DNA

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5
Q

stAGE 3 OF pcr (Elongation)

A

The reaction mixture is now heated to 72oC, (optimum for DNA polymerase).
Taq polymerase forms phosphodiester bonds between DNA nucleotides alongside each template strand. Complementary strands of DNA are built up by complementary base pairing.

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6
Q

_____ new copies of the fragment of DNA are formed in the first cycle of PCR.

A

Two

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7
Q

The cycle starts again with all _____strands being used (two original and two new). Therefore, the amount of DNA ______ with each cycle.

A

four

doubles with each cycle.

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8
Q

Reaction mixture is set up containing:

A

Reaction mixture is set up containing the DNA sample, buffer solution, free nucleotides, primers (short pieces of DNA complementary to the bases at the start of the fragment) and DNA polymerase (taq polymerase)

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9
Q

What do restriction enzymes do?

A

recognise specific palindromic sequences (aka recognition sequences) and cut the DNA at these places `

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10
Q

Example of restrition enzymes

A

EcoRI cuts at GAATTC

HindIII cuts at AAGCTT

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11
Q

what is a palindromic sequence

A

consist of antiparallel base pairs (base PAIRS that read the same in opposite direction

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12
Q

what is another name for a restriction enzyme

A

Restriction endonucleases

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13
Q

What is a sticky end

A

Unpaired (single stranded DNA) bases at either end of the cut fragment

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14
Q

why are sticky ends useful

A

Allow DNA to anneal with other piece of DNA

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15
Q

Why might you have to incubate DNA with two different restriction enzymes?

A

If desired fragment has different restriction sites either side of it

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16
Q

DNA sequencing

A

technique where we can map out the base sequence of an individuals genome

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17
Q

another name for chain termination method

A

Sanger method

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18
Q

chain termination method

Process

A

1)DNA can be sequenced using the chain termination method.
2)Four tubes are needed which contain the single stranded DNA template, DNA polymerase, DNA primers and free nucleotides (A, T, C and G).
3)Each separate tube also contains a different fluorescently tagged nucleotide (A, T C or G). If this is added to the DNA chain, no other bases can be added after it.
4)All the tubes go through PCR.
5)This produces lots of DNA strands of different lengths, because the fluorescently tagged bases were added at different points.
6)The DNA fragments are separated by electrophoresis.
7)The gel is viewed under a UV light and the complementary base sequence can be read from the gel.
8)The smallest polynucleotide travels the furthest, so the sequence is read from the bottom of the gel to the top.

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19
Q

problems with chain termination method

A

(-) slow- can only sequence fragments shorter than 1000 base pairs (usually 750)
(-) Expensive, would have to split up longer sequences and run them many times

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20
Q

Automated DNA sequencing

A

The chain termination method has now become automated and faster.
Tubes are still used that contain the modified nucleotides, each with a different coloured fluorescent label but a machine now reads the sequence instead of running the gel through electrophoresis.

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21
Q

Advances in DNA sequencing- High throughput sequencing

A

Millions of fragments of DNA are amplified, sequenced and imaged at the same time ( in parallel) so it is much faster to sequence DNA
This has massively reduced the cost of sequencing large amounts of DNA
Use state of the art computer technology

22
Q

Genomics Definition

A

Genomics is the study of whole sets of genetic information. It involves sequencing DNA bases that occur in the cells of a particular species.

23
Q

Genome sequencing process

A

1)A genome is cut into smaller segments (about 100,00bp) using restriction enzymes.
2)Each fragment is inserted into a different BACs (man-made plasmids).
3)Each BAC is inserted into a different bacteria.
4)The bacteria divide creating colonies of clones which contain a specific DNA fragment these make a complete genomic DNA library.
5)DNA is extracted from each colony and cut up using restriction enzymes to produce overlapping pieces of DNA.
6)Each piece of DNA is sequenced using chain termination method.
7)The pieces are put back in order to give the full sequence from that BAC.
8)Computers are used to put all the DNA fragments from all the BACs in order. This completes the genome.

24
Q

Computational biology-

A

using computers to study biology e.g to create computer simulations & mathematical models

25
Q

Bioinformatics

A
  • developing and using computer software that can analyse, organise and store biological data
26
Q

Synthetic biology

A
  • A large field of biology that includes building biological systems from artificially- made molecules, redesigning biological systems to perform better and designing new biological systems/molecules that don’t exist naturally.
27
Q

why is synthetic biology useful

A

Produce energy + drug products
e.g Anti malarial drug: Artemisinin

28
Q

description of studying genotype-phenotype relationships

A

-Predict an organisms phenotype by analysing it’s genotype

29
Q

uses of studying genotype-phenotype relationships

A

-Predict what health problems a person is likely to face

30
Q

description of Epidemiological studies

A

study of health and disease within a population.

considers distribution of a disease, its cause and effects

31
Q

uses of epidemiological studies

A

computerised comparisons between genomes of people who have a disease and those that don’t can be used to detect particular mutations that could be responsible for the increased risk of disease

32
Q

sequencing genomes on Understanding evolutionary relationships

A

whole genomes of different species analysed using computer software to tell us how closely related different species are. Tell us ab evolutionary relationships

33
Q

uses of understanding evolutionary relationships

A

Build up a picture of early human migration

34
Q

what is gel electrophoresis

A

A procedure that uses electricity to separate out DNA fragments, RNA fragments or proteins depending on their size.

35
Q

electrophoresis process

A

Agarose gel with a row of wells at one end.

Micropipette used to add a set volume of DNA and loading dye to the wells.

Gel box containing buffer solution surface of gel tray is covered in buffer solution which carries charge to separate fragments

Gel tray added to gel box, lid added and connected to power supply. Turned on for 30 minutes which causes an electrical current to pass through gel towards anode.

DNA fragments are negatively charged.
End of the gel tray with the wells must be closest to negative electrode(cathode).

Gel tray is removed and buffer solution removed.

Staining solution is added to gel tray to stain DNA fragments. Gel tray is then rinsed with water.

Smallest DNA fragments move furthest away from wells towards anode.

36
Q

Precautions when conducting gel electrophoresis

A

Micropipette must not touch bottom and must have a clean tip each time.

Gloves should be worn.

37
Q

what does loading dye do (gel electrophoresis

A

Loading dye helps samples to sink and makes them easier to see

38
Q

DNA profiling

A

Some of an organism’s genome consists of repetitive non-coding base sequences.
The number of times these are repeated varies from person to person.
The number of times a sequence is repeated at specific loci can be analysed using gel electrophoresis which creates a DNA profile.

39
Q

2 uses of DNA profiling

A

-Forensic use
-Medical use

40
Q

uses of DNA profiling in Forensic science

A

DNA collected at a crime scene can be isolated then amplified using PCR. This is then run through electrophoresis gel and can be compared to DNA profiles of suspects.

41
Q

uses of DNA profiling in Medical diagnosis

A

Can make a DNA profile of several alleles which can be used to analyse the risk of genetic disorders  can be used when a specific mutation isn’t known or where several mutations could have caused the disorder.

42
Q

pyrosequencing 6 steps

A

1)section of DNA is cut into fragnments, split into single strands and then a strand from each fragment is attached to a small bead

2) PCR is used to asmplify the aDNA fragments on each bead

3)Then each bead is put into a seperate well

4) The 4 different types of nucleotides added to the wells one after the other, over and over for 100 cycles. The Free nucleotides added to wells attach to DNA strands via complementary base pairing.

5) Wells also contain specific enzyme which cause light to be emitted when a nucleotide is added to the DNA strand.
(More than one nucleotide can be added at a time if bases are the same, so intensity of light can vary)

6) Computers analyse the occurrence and intensities of the light emitted in the different wells, after each type of nucleotide is added, and process this info to interpret the DNA sequence

43
Q

Describe how DNA can be visualised after electrophoresis has been completed

A

-radioactive labels/tags
-fluorescent labels/tags
-UV light/radiation
-Visible stain

44
Q

If a DNA sequencing technique has an error probability of 1 in 100 nucleotides,

If a protein is 200 amino acids in length, How many errors are expected in the exons of the sequenced gene

A

6

because each 1 amino acid is coded for by 3 bases.

3x200 = 600

600x 1/100 = 6

45
Q

explain why only selected sections of non-coding DNA are used when profiling a human (3mrks)

A

in most people, the genome is very similar/most genes are the same (1)

using coding sequences would not provide unique profiles (1)

non-coding DNA contains variable numbers of repeating sequences (1)

46
Q

suggest why a mutation may lead to the reduction of effectiveness of a drug that binds to protein receptors in the lining of the bronchioles

A

-Change in base sequence = change in amino acid sequence/primary structure (1)

-= Change in tertiary structure of binding site of receptor (1)

-Drug unable to bind (1)

-No response triggered (1)

47
Q

state one development other than nanopore technology that has led to an increase in the speed at which DNA can be sequenced

A

-Pyrosequencing
- High throughput sequencing

-massive parallel sequencing
-WGS
-NGS
-Shotgun sequencing

48
Q

Differences between G nucleotide and ATP

A

G contains guanine, ATP contains adenine

G contains deoxyribose, ATP contains ribose

G has 1 phosphate (not phosphate molecule!!), ATP has 3 phosphates

G has phosphate attached to C3 , ATP has no phosphate attached to C3

49
Q

explain how DNA sequencing allows the swquence of amino acids in a polypedptide chain to be predicted

A

-sequence/order, of bases codes for order of amino acids/polypeptide sequence

-Triplet codes for one amino acid

50
Q

Outline how DNA sequencing and bioinformatics could be used to increase the effectiveness of a vaccination program against Ebola

A

(sequencing)
1) (high) mutation (rate) means many strains of virus exist
2) Can predict strain/protein/antigen
3) So vaccine contains antigen

(Bioinformatics)
4) facilitates access to large amount of data
5)Facilitates access to data on DNA and proteins
6) format of information is universal

7) can identify source of outbreak
8)Can identify vulnerable populations
9) Vaccination program can target certain area / individuals

biology (45 decks)

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