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70 genetics > L8 - molecular bio techniques - sequencing&PCR > Flashcards

L8 - molecular bio techniques - sequencing&PCR Flashcards

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1
Q

what is DNA sequencing?

A

producing the exact sequence of bases within DNA clones

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2
Q

why is DNA sequencing useful

A
  1. can predict AA sequence of protein the gene encodes
  2. gives info on genome organisation
    - regulatory regions
    - telomers
    - centromeres
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3
Q

why are ddNTPs used in DNA sequencing

A

because they don’t contain an OH on C3 so DNA pol cant incorporate more nucleotides after a ddNTP

therefore cause chain termination

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4
Q

describe process of DNA sequencing (sanger ddNTPs)

A
  1. small amount of radiolabelled ddCTP, dCTP, all other nucleotides, primer and DNA pol added
  2. wherever a ddCTP is incorporated it will result in fragments of various sizes that can be detected as they’re radiolabelled
  3. repeat with ddNTPs of G / A / T
  4. run each separately on polyacrylamide gel which will separate fragments differing by single nucleotide
  5. expose to x-ray film to detect radioactivity
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5
Q

describe process of automated DNA sequencing

A
  1. single reaction where all ddNTPs are added but each is labelled with a unique fluorescent marker
  2. colour at end of fragment identified
  3. fragments separated on capillary gel
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6
Q

what gel used in sanger DNA sequencing

A

polyacrylamide

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7
Q

what gel used in automated DNA sequencing

A

capillary gel

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8
Q

how is fluorescence detected in automated sequencing?

A

each fluorescent tag has a different wavelength

as the fragments move down the gel they excite a laser

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9
Q

uses of DNA sequencing

A
  1. study disease
  2. study evolution
  3. study individual differences (SNPs)
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10
Q

what are SNPs?

A

single nucleotide polymorphisms

differences in single base pairs between individuals

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11
Q

what are the non protein coding regions of genome responsible for?

A
  1. regulatory regions
  2. RNA
  3. introns
  4. repetitive DNA
  5. UTRs
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12
Q

what is bioinformatics?

A

computer techniques to organise share + analyse the large amounts of sequencing info

interactive databases to store info
mathematics & statistics to analyse info

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13
Q

what is structural and functional genomics

A
  1. identifying features in genome
  2. identifying gene structure
  3. predict regions eg regulatory, promoter, protein coding etc
  4. predict gene functions
  5. identify gene clusters & families
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14
Q

what is comparative genomics

A
  1. comparing genomics between species
  2. looking at conserved sequences (synteny)
  3. evolution of genes
  4. relationship between genomes and organisms environment
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15
Q

what is PCR

A

selective amplification of specific regions of DNA

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16
Q

what type of polymerase is used in PCR and why

A

taq polymerase

thermostable - can be used at high temp

17
Q

briefly describe overall method of PCR

A

two primers added either end of the region wanted to be copied, taq polymerase will copy the DNA between the two

18
Q

what is the smallest amount of DNA that can be used in PCR

A

10^-15

19
Q

uses of PCR

A
  1. forensics
  2. contamination in food
  3. study of disease
  4. paternity testing
20
Q

what are the steps of PCR in order

A

denaturation
primer annealing
extension

21
Q

describe what happens in denaturation process of PCR

A

heated to 95 to denature DNA to single strands

22
Q

what is the name of the machine that completes PCR

A

thermocycler

23
Q

describe what happens in primer annealing process of PCR

A

temp reduced to 45-68 to allow forward and reverse primers to bind (to each strand)

24
Q

describe what happens in primer extension process of PCR

A

temp 72

taq synthesises DNA between primers (of each strand)

25
Q

list some limitations of PCR

A
  1. info of nucleotide sequence needs to be known in order to bind primers
  2. very sensitive, minor contamination may cause major problems
  3. cant amplify long segments (up to 8kb)
26
Q

what is reverse transcription PCR

A

studying gene expression by examining mRNA (using cDNA) in certain tissues

27
Q

what is quantitative real time PCR

A

allows researchers to quantify amplification reactions as they occur in real time

70 genetics (19 decks)

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