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NUT1004 > Molecular techniques > Flashcards

Molecular techniques Flashcards

1
Q

How can the sample affect the DNA/RNA purification method

A

Some tissues are easy to access with high RNA/DNA content, some tissues have high protein/fat content, some DNA sources affect the quality of DNA/RNA (faecal and urine samples)

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2
Q

What are the 5 major steps in studying DNA and RNA

A

Sample selection, extraction/purification, amplification, analysis, interpretation

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3
Q

Why is sample selection of DNA and RNA important (2 reasons)

A

DNA/RNA can vary between tissues and the sample can affect the extraction/purification method

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4
Q

Why is DNA extraction and purification important

A

DNA must be isolated and purified from source material contaminants e.g. RNA, proteins, fats

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5
Q

What is the DNA extraction/purification steps (5)

A

Break up source, break open cells, separate DNA, purify DNA, stock for analysis

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6
Q

What is Isolation/amplification of a target extracted DNA sample

A

Analysis off a particular part of DNA by PCR and visualisation, quantitative PCR and cloning

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7
Q

What is PCR

A

The amplification of a specific region/gene of DNA from the DNA pool

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8
Q

What is included in the PCR mix

A

Template, primer, nucleotides, polymerase, buffer

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9
Q

What happens in the denaturation stage of PCR

A

DNA template strands are opened

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10
Q

What happens in the annealing stage of PCR

A

Primers bind to region of interest

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11
Q

What happens in the extension stage of PCR

A

Region of interest is copied (polymerised)

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12
Q

Why does PCR have an exponential increase

A

Copies of DNA doubles each time until limited by nucleotides

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13
Q

At what temperature does denaturation occur in PCR

A

94

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14
Q

At what temperature does priming occur in PCR

A

55 >68

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15
Q

At what temperature does extension occur in PCR

A

72

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16
Q

What is DNA visulisation

A

Visualisation of stained DNA through a gel matrix where DNA is separated by size and pulled through a porous medium (agarose) by an electrochemical gradient

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17
Q

What does almost every DNA/RNA based technique begin with

A

PCR

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18
Q

What is PCR used for

A

Preparation of DNA for sequencing for medical diagnosis, DNA fingerprinting and paternity testing

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19
Q

When is quantitative PCR used

A

If we want to measure the amount of nucleic acid in given sample

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20
Q

What is quantitative PCR

A

Extremely sensitive real time monitoring of PCR amplification using fluorescent dyes which counts DNA molecules/measures copies of DNA

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21
Q

How is DNA quantity assessed in quantitative PCR

A

As reaction progresses using a DNA labelling dye,

22
Q

What are the practical applications of quantitative/ qPCR

A

Anything where DNA abundance is of interest e.g. copy number variation to measure the amount of a specific DNA molecule such as to detect chromosomal abnormalities

23
Q

How is the effect of mutation or a specific gene studied

A

DNA cloning

24
Q

What is DNA cloning

A

Recombinant DNA technology

25
Q

What are the 3 steps of DNA cloning

A

1) DNA target is inserted into plasmid
2) plasmid is transferred into a host organism
3) host organism replicates the target DNA

26
Q

What is a plasmid

A

Small piece of DNA that contains the genes necessary to replicate or express the DNA target

27
Q

What are the 3 practical applications of DNA cloning

A

Biological factory for bulk production of DNA/RNA/protein
Study of the expression of a target in other organisms to understand it’s function (transgenic)
To give an organism a new trait/feature e.g resistance crops

28
Q

What 4 processes are involved in analysis of extracted/purified and amplified DNA

A

Sanger sequencing, gene expression, metagenomics and next generation sequencing

29
Q

What is DNA sequencing

A

Visualisation and determination of DNA sequence, analysis of exact sequence of DNA you’re observing to detect variations using chain termination

30
Q

Describe DNA sequencing

A

standard PCR mix and 2 types of nucleotide (DNTPs and diDNTPs) are used to synthesise every possible length of PCR product with a dye label as when diDNTP is incorporated (labelled nucleotide) the chain is terminated

31
Q

What is the difference between Deoxyribosenucelotides and Di-deoxyribosenucelotides

A

DNTPS can bind to the next base pair in the DNA chain, diDNTPS are dye labelled and cannot bind to the next base pair in DNA as have a H instead of OH group on C3

32
Q

What are the practical applications of DNA sequencing

A

Diagnostic testing for disease, forensics, paternity testing, public health/viral and infection tracing

33
Q

How are the products of DNA studied (what is expressed)

A

RNA arrays ans RNAseq

34
Q

What is a micro array

A

Simultaneous analysis of thousands of genes in one experiment

35
Q

What are the 4 steps of a micro array

A

1) RNA is extracted and converted to stable cDNA
2) cDNA is loaded onto an array with many probes for different genes
3) cDNA can bind to each probe
4) Successful binding release a fluorescent signal, signal intensity =amount of cDNA/RNA (genes are present in sample and can be quantified)

36
Q

What is RNA sequencing

A

Alternative to array expression analysis to record the RNA sequence and amount in a sample

37
Q

What are the 4 steps of RNA sequencing

A

1) RNA is extracted and converted to cDNA
2) cDNA molecules are fragmented into smaller parts and sequenced many times
3) Each fragments is aligned to a reference
4) amount of fragment is equal to amount of RNA in sample

38
Q

What are the practical applications of arrays and RNAseq

A

Study of transcriptomics (assigning function to genetic variation), studying the effect of a mutation on different cells/tissues

39
Q

What is next generation sequencing

A

The study of all DNA in a sample at the same time

40
Q

What is whole genome sequencing

A

Sequencing of introns and exons

41
Q

What is whole exome sequencing

A

Exons only

42
Q

Describe the 4 steps of next generation sequencing

A

1) DNA is sheared into smaller fragments
2) exaggerated fragment is sequenced (only exons in whole exome sequencing using probes and arrays)
3) software aligns each fragment to a reference version of genome
4) comparisons are made between sample and reference to identify changes

43
Q

What are the practical applications of next generation sequencing

A

Diagnostic testing for disease (unbiased as all genes), novel gene identification, personalised medicine

44
Q

What is the difference between NGS and DNA sequencing

A

NGS is faster, on a larger scale and more economical

45
Q

What is Metabiomics

A

Studying DNA that isn’t ours (microbiomics)

46
Q

What is metabiomics used for

A

To study the DNA of the gut microbiome

47
Q

What type of sequencing is used in metabiomics

A

Next generation sequencing

48
Q

What 4 steps are involved in metabiomics

A

1) sample microbes from source (skin, gut etc.)
2) extract DNA and sequence by NGS to determine Volans number of different microbes in sample
3) microbial abundance= sequence amount
4) analysis

49
Q

What are the two types of analysis used to metabiomics

A

Taxonomy profiling and functional profiling

50
Q

What is taxonomy profiling

A

Comparing what should be present to what is present

51
Q

What is functional profiling

A

Comparing what is there under different conditions e.g, diet changes, disease

52
Q

What are the practical applications of metabiomics/microbiomics

A

Microbiome in human health, disease, infection, drug discovery, agriculture, agrifood, environment, veterinary

NUT1004 (14 decks)

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