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Medical Cell Biology and Genetics > L27- Advanced molecular techniques > Flashcards

L27- Advanced molecular techniques Flashcards

1
Q

different molecular techniques must be employed when looking at

A

micro and macromutations

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2
Q

analysis of DNA can occur at

A
  • nucleotide level
  • gene level
  • chromosome level
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3
Q

1.Analysis of DNA at nucleotide level

A

DNA sequencing

PCR plus restriction analysis/ DNA sequencing

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4
Q

Analysis of DNA at the gene level

A
  • Southern hybridisation
  • Northern hybridisation
  • RT PCR
  • Microarray
  • DNA fingerprinting
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5
Q

Analysis of DNA at the chromosome level

A
  • Karyotyping
  • FISH/ Chromosome painting
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6
Q

DNA sequencing most famous method

A

sanger sequencing method

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7
Q

Sanger chain termination

A

dideoxy chain termintation

  • allows us to work out the nucleotide sequence of a piece fo DNA (able to find micro-mutations)
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8
Q
A
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9
Q

sanger chain termination method

A
  1. Mixture of dNTPs added
  2. Deoxynucleotide triphosphate molecules (dNTP) which can be used in DNA replication (normal 3’ hydroxyl group which allows DNA polymerase to add another dNTP)
  3. Then one at a time different dideoxynucleotide triphosphate (ddNTP- e.g. dATP, dCTP, ddGTP or ddTTP), which contain a H at the 3’ position, are added
    1. DNA polymerase can use as a substrate, adding it to the growing DNA polymer, it cannot elongate on from it (no more subsequent phosphodiester bonds will form with new dNTPs)
  4. 4 separate tubes are used each with a unique ddNTP
  5. e.g. in one tube there will be dNTPs, DNA polymerase, labelled primer and ddCTP
  6. Incubate at 370C what do we see? Wherever C needs to be added in the sequence there is a chance that:
    • i) A dCTP will be used and the chain will continue growing
    • ii) A ddCTP will be used and stop further growth
  7. Because we have lots of the template, overtime we will see a mixture of new DNA molecule produced of different lengths depending on where the ddCTP is incorporated
  8. After incubation the products of the reaction are run out on a gel sing separate lanes for each reaction tubes
  9. This will separate the labelled fragments on the basis of size
  10. We are then able read off the sequence from the bottom of the gel to work out the nucleotide sequence in the newly synthesised strand
  11. The sequence can then be interpreted e.g. ATGCCTGCA
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10
Q

difference betweend deoxynucleotide triphosphoate (dNTP) and dideoxynucleotide triphosphate (ddNTP)

A

deoxynucleotide triphosphoate (dNTP) - which can be used in DNA replication (normal 3’ hydroxyl group which allows DNA polymerase to add another dNTP)

dideoxynucleotide triphosphate (ddNTP- e.g. dATP, dCTP, ddGTP or ddTTP) contain a H at the 3’ position

DNA polymerase can use as a substrate, adding it to the growing DNA polymer, it cannot elongate on from it (no more subsequent phosphodiester bonds will form with new dNTPs)

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11
Q

using genome sequencing we can look at

A

other animal genomes to learn about human disease

e. g. the fact gorillas have very similar genome, but are resistant to malaria
- could be used to develop strategies for humans

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12
Q

DNA sequencing ethical considerations

A

Who would be interested in your genome info?

  • Family
  • Potential spouse
  • Doctors
  • Government
  • Police
  • Schools
  • Insurance company

Can the knowledge help prevent illness in later life?

Does it open up areas for discrimination?

Who owns DNA sequence?

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13
Q

PCR

A

Amplifies specific segments of DNA

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14
Q

What is needed for PCR?

A

1) Primers
2) dNTPs (free nucleotides)
3) Taq polymerase
4) DNA template to be copied
5) Buffer
6) Thermocycler

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15
Q

PCR Process

A

1) Denaturing (90-95)- separate double strands
2) Annealing (50-56)- temp lowered to enable primers to bind
3) Extension (72)- temp raised and new strands synthesised by polymerase

–> Cycle repeated- amount of DNA doubles every time

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16
Q

PCR uses what sort of primers

A

allele- specific primers- isolates point mutations

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17
Q

allele- specific primers

A

In this approach, the specific primers are designed to permit amplification by DNA polymerase only if the nucleotide at the 3’-end of the primer perfectly complements the base at the variant or wild-type sequences. After the PCR and electrophoresis, the patterns of specific PCR products permit the differentiation of the SNPs.

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18
Q

Southern blotting uses

A

uses DNA probes to identify complementary DNA sequences after gel electrophoresis

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19
Q

Northern blotting

A

uses DNA to detect RNA after gel electrophoresis

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20
Q

western uses

A

not a DNA hybridisation technique. Involves detection of proteins by antibodies after protein gel electrophoresis

21
Q

why use soutehrn hydridisation

A

Investigate gene structure e.g. large deletions or duplications

To investigate gene expansions such as triplet repeats e.g. Huntington’s

To investigate mutations in genetics tests

To investigate variation, genetic relationships e.g. DNA fingerprinting

22
Q

southern hydridiation allows

A

visualistion of specific DNA e.g. to see if individual has specific DNA that codes for a disease

23
Q

southern hydridisation process

A

Take DNA sample and cleave using endonucleases à smaller pieces of DNA

DNA fragments are run on a gel electrophoresis which separates fragments based on charge and size

Take gel electrophoresis and transfer onto nitrocellulose filter (place on top and the fragments will transfer absorb onto the filter)

Take the filter and expose to radiolabelled / fluorescently labelled probe (ssDNA complementary to gene of interest)- hybridisation

Probe will anneal to gene of interest which has complementary DNA

Then expose filter to x-ray (radiolabelled)- will expose if gene A is present or not

24
Q

probes in blotttingare either

A

Radiolabelled or fluorescently labelled

25
Q

probes do not have to have

A

100% similarity to the target sequence

26
Q

when probs have 100% similarity

A

binds tightly

27
Q

when probes have 80% similarity

A

bind but less tightly

28
Q

probes also do not have to

A

span the whole range of the target gene

29
Q

probes will not affect

A

the position of the target sequence on a gel

30
Q

Northern hydridisation

A

Similar process to southern blotting, however runs RNA instead of DNA on a gel electrophoresis – could use mRNA from cytoplasm

31
Q

RT- PCR

A

RNA is converted to cDNA by reverse transcriptase (RT), and then the cDNA is amplified by PCR.

32
Q

application of RT-PCR

A
  1. Detection of expressed genes
  2. Examination of transcript variants
  3. Generation of cDNA templates for cloning and sequencing
33
Q

RT- PCR process

A

RNA consists of start codon AUG

Oligonucleotide primer (dT) primer added which binds to RNA poly A tail

RT and dNTPs added

RT binds to dT primer and synthesises cDNA by adding dNTPs in the 5’ to 3’ direction

cDNA amplified using Taq polymerase during PCR

34
Q

what part of DNA does DNA fingerprinting use

A

Uses parts of non-coding regions to profile the DNA of different individuals.

35
Q

why does DNA fingerprinting use DNA fingerprinting

A

due to high variability between individuals compared to genes

  • like short tandem repeats (STR)
36
Q

STR also refered to as

A

minisatellites

37
Q

in three related individuals there genes will be

A

the same, but their STR will be different

  • e..g different number of STR
38
Q

How does DNA fingerprinting work

A

By removing STR (minisatellites) using restriction endonucleases

Amplifying them using PCR

Separating them based on their size using gel electrophoresis

39
Q

How can DNA fingerprinting be used

A

can be used to detect murderers, show family relationships (paternal)

40
Q

microarray

A

compares gene expression in 2 conditions

41
Q

outline broad icroarray technique

A
  1. mRNA extracted from both healthy and diseased cell (10,000s of genes- genome wide)
  2. mRNA reverse transcribed into more stable cDNA
  3. Disease cells labelled red and healthy sample labelled green
  4. Both labelled samples then combined
  5. Sample then added to a microarray chip
  6. Labelled cDNA from sample bind to complementary DNA in the microarray
  7. Bound cDNA will bind tightly to its complementary DNA (hybridisation) on the chip, all unbound cDNA will be rinsed off
  8. Then chip put into electronic scanner, which activates fluorescent die
  9. Calculates the ratio of red to green DNA in each well, indicating which genes are expressed in the normal and diseased cell
42
Q

microarray can be used to show

A

E.g. which genes are activated or repressed during disease

43
Q

two types of microarray

A

Conditional gene expression microarray

Array comparative genome hydridisation

44
Q

Conditional gene expression microarray

A

Compare cells (cancer vs normal cells) from the same individual

45
Q

array comparative genome hydridiation

A

Extracts and labels DNA from cells form a normal individual and from an individual with a specific disease.Evaluates areas of the human genome for gains or losses of chromosome segments at a higher resolution than traditional karyotyping.

46
Q

karyotyping

A

Cytogenetics, involving analysis of the entire chromosome complement through the microscope.

Evaluates:

Chromosome number

Chromosome structure

47
Q

FISh

A

fluorescent in situ hydridisation

48
Q

fluorescent in situ hydridisation

A

Uses fluorescent probes that binds to only those parts of the chromosome with a high degree of sequence complementarity. It is used to detect and localise the presence or absence of specific DNA sequences on chromosomes

à Fluorescent microscopy can then be used to find out where the fluorescent probe is bound to the chromosome (can be visualised)

Important tool for:

Understanding a variety of chromosomal abnormalities and other genetic mutation

49
Q

FISH process

A
  1. Make a probe complementary to the know sequence
  2. When making the probe label it with a fluorescent marker
  3. Put the chromosomes on a microscope slide and denature them
  4. Denature the probe and add it to the microscope slide, allowing the probe to hybridise to its complementary site
  5. Wash off excess probe and observe the chromosome under fluorescent microscope

Medical Cell Biology and Genetics (33 decks)

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