L8 - molecular bio techniques - sequencing&PCR Flashcards
what is DNA sequencing?
producing the exact sequence of bases within DNA clones
why is DNA sequencing useful
- can predict AA sequence of protein the gene encodes
- gives info on genome organisation
- regulatory regions
- telomers
- centromeres
why are ddNTPs used in DNA sequencing
because they don’t contain an OH on C3 so DNA pol cant incorporate more nucleotides after a ddNTP
therefore cause chain termination
describe process of DNA sequencing (sanger ddNTPs)
- small amount of radiolabelled ddCTP, dCTP, all other nucleotides, primer and DNA pol added
- wherever a ddCTP is incorporated it will result in fragments of various sizes that can be detected as they’re radiolabelled
- repeat with ddNTPs of G / A / T
- run each separately on polyacrylamide gel which will separate fragments differing by single nucleotide
- expose to x-ray film to detect radioactivity
describe process of automated DNA sequencing
- single reaction where all ddNTPs are added but each is labelled with a unique fluorescent marker
- colour at end of fragment identified
- fragments separated on capillary gel
what gel used in sanger DNA sequencing
polyacrylamide
what gel used in automated DNA sequencing
capillary gel
how is fluorescence detected in automated sequencing?
each fluorescent tag has a different wavelength
as the fragments move down the gel they excite a laser
uses of DNA sequencing
- study disease
- study evolution
- study individual differences (SNPs)
what are SNPs?
single nucleotide polymorphisms
differences in single base pairs between individuals
what are the non protein coding regions of genome responsible for?
- regulatory regions
- RNA
- introns
- repetitive DNA
- UTRs
what is bioinformatics?
computer techniques to organise share + analyse the large amounts of sequencing info
interactive databases to store info
mathematics & statistics to analyse info
what is structural and functional genomics
- identifying features in genome
- identifying gene structure
- predict regions eg regulatory, promoter, protein coding etc
- predict gene functions
- identify gene clusters & families
what is comparative genomics
- comparing genomics between species
- looking at conserved sequences (synteny)
- evolution of genes
- relationship between genomes and organisms environment
what is PCR
selective amplification of specific regions of DNA
what type of polymerase is used in PCR and why
taq polymerase
thermostable - can be used at high temp
briefly describe overall method of PCR
two primers added either end of the region wanted to be copied, taq polymerase will copy the DNA between the two
what is the smallest amount of DNA that can be used in PCR
10^-15
uses of PCR
- forensics
- contamination in food
- study of disease
- paternity testing
what are the steps of PCR in order
denaturation
primer annealing
extension
describe what happens in denaturation process of PCR
heated to 95 to denature DNA to single strands
what is the name of the machine that completes PCR
thermocycler
describe what happens in primer annealing process of PCR
temp reduced to 45-68 to allow forward and reverse primers to bind (to each strand)
describe what happens in primer extension process of PCR
temp 72
taq synthesises DNA between primers (of each strand)
list some limitations of PCR
- info of nucleotide sequence needs to be known in order to bind primers
- very sensitive, minor contamination may cause major problems
- cant amplify long segments (up to 8kb)
what is reverse transcription PCR
studying gene expression by examining mRNA (using cDNA) in certain tissues
what is quantitative real time PCR
allows researchers to quantify amplification reactions as they occur in real time
