lecture 2 Practical applications of molecular biology 1.1 Flashcards
what is a POLYMERASE CHAIN REACTION (PCR)
*PCR is a commonly used approach to detect the presence or absence of a specific DNA sequence (or gene), e.g. an antibiotic resistance gene.
*Gene-specific primers are used to amplify the sequence of interest during the PCR reaction
how is PCR based on DNA replication
*Template DNA – the original DNA molecule that needs copying (only trace amounts suffice).
*DNA primers - short pieces of single-stranded DNA (aka oligonucleotides) that match the sequences at either end of the target DNA segment.
*Deoxynucleoside triphosphates (dNTPs)- building blocks.
*DNA polymerase -enzyme
what does a Polymerase Chain Reaction need
*The PCR process requires cycling through several different temperatures - PCR machines are sometimes called thermocyclers
Heat to melt DNA
Cool to anneal DNA primers
Extend primers using DNA polymerase
what can PCR amplify?
PCR can be used to amplify a specific DNA sequence
what is the end result of PCR?
End result of PCR is an amplified targe
what do repeated PCR cycles do?
Repeated cycles increase number of amplified DNA by 2n
what does a thermocycler do?
Thermocycler is used to amplify DNA
Recurring temperature increase will denature DNA polymerase
thermostable DNA polymerase makes PCR a fully-automated process
*Taq DNA polymerase originally from Thermus aquaticus catalyses reaction at 72oC and survives denaturation at 95oC.
*A thermostable DNA polymerase means there is no need to replenish the DNA polymerase after each cycle the process becomes fully automated.
what Influence does temperature have on correct primer binding
Optimum annealing temperature is required to ensure specific binding of primers.
(a)Too high –> no PCR product
(b)Too low –> too many non-specific PCR products
(c)Optimum temp –> desired PCR product
how can PCR be used to obtain genomic clones
*Total genomic DNA is first purified from cells.
*PCR is used to clone a segment of chromosomal DNA.
*PCR primers that flank the stretch of DNA to be cloned are added, and many cycles of PCR are completed.
*Because only the DNA between (and including) the primers is amplified, PCR provides a way to obtain selectively any short stretch of chromosomal DNA in an effectively pure form.
what does GEL ELECTROPHORESIS do?
*Gel electrophoresis separates DNA molecules of different sizes.
*The amplified products (or amplicons) from a PCR reaction can be separated by performing gel electrophoresis and visualised as ‘bands’ following the addition of DNA binding dyes to the agarose gel and exposure to UV illumination.
*This allows their size/length to be compared with what you expected to get, i.e. the length of the target gene including the forward and reverse primers
what is DNA SEQUENCING
*Changes to an organism’s genotype can be seen using PCR and DNA sequence analysis.
*PCR may also be used to detect the presence or absence of specific genes, e.g. those encoding antibiotic resistance, and their sequence compared to a database to find the closest match.
*DNA can be sequenced rapidly by dideoxy sequencing.
why are ddNTPs important?
*The dideoxy method of sequencing DNA relies on chain-terminating dideoxyribonucleoside triphosphates (ddNTPs)
*These ddNTPs are derivatives of the normal deoxyribonucleoside triphosphates (dNTPs) that lack the 3 -′hydroxyl group.
*When incorporated into a growing DNA strand, they block further elongation of that stran
what does each coloured peak represent?
Each colored peak represents a nucleotide in the DNA sequence
how have So-called next-generation sequencing (NGS) methods have revolutionised DNA analysis
*A genome or other large DNA sample is broken into millions of short fragments.
*The fragments are attached to the surface of a flow cell and amplified by PCR to generate DNA clusters, each containing about a 1000 copies of a single fragment.
*In STEP 1, the anchored DNA clusters are incubated with DNA polymerase and a special set of all four nucleoside triphosphates (NTPs), each with two reversible chemical modifications: a uniquely colored fluorescent marker and a 3 chemical group that terminates DNA synthesis. ′Normal dNTPs are not present.
*After a nucleotide is added by DNA polymerase, a high-resolution digital camera records the color of the fluorescence at each DNA cluster
