IMMUNOLOGIC AND MOLECULAR DIAGNOSTICS II Flashcards
Molecular-based diagnostic methods are notably based on the processes of the
We copy or modify these processes to execute them in vitro.
Central Dogma of Molecular Biology
Central Dogma of Molecular Biology processes
replication, transcription and translation
Major Stages in DNA Replication
Formation of the Replication Fork
Primer binding
Elongation (DNA polymerase)
Termination
a technique that amplifies a specific DNA segment into billions of copies, enabling scientists to study a small DNA sample in detail.
does not have the same enzymes as the naturally occuring dna replication
POLYMERASE CHAIN REACTION (PCR)
POLYMERASE CHAIN REACTION (PCR) parts
dna template
primers
Taq polymerase
dNDPs
Buffer/Cofactors
POLYMERASE CHAIN REACTION (PCR) parts
Short DNA sequences that serve as the starting point of the amplification.
Determines w/c portion of the genome is to be sequenced.
Specificity of the PCR process depends on its design
primers
POLYMERASE CHAIN REACTION (PCR) parts
is a heat-stable version of the DNA pol enzyme used
Taq polymerase
enzymes that need Mg2+ as cofactors
Polymerases
END-POINT ANALYSIS OF PCR types
gel electrophoresis, NA hybridization (blotting) and Sequencing, Northern Blot (RNA), Southern Blot (DNA)
Sanger Sequencing
POLYMERASE CHAIN REACTION (PCR) process
Sample of the project
DNA extraction
Thermal cycler
Gel Electrophoresis (or other END-POINT ANALYSIS OF PCR)
DNA sequencing
Thermal cycler temperature for
denaturation and DNA separation fork
94
Thermal cycler temperature for initial denaturation
94
Thermal cycler temperature for annealing (attaches primers)
50-65
Thermal cycler temperature for extension. 1 min per kilobasepair
72
Thermal cycler temperature for elongation polymerize
72
END-POINT ANALYSIS OF PCR
a laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size. the molecules to be separated are pushed by an electrical field through a gel that contains small pores.
Gel Electrophoresis
Gel Electrophoresis process
PCR DNA amplicons on agarose gel
Eletric current
DNA separation
UV transillumination and documentation
DNA amplicaons show up in bands
DNA ladder will be used as reference
POSITIVE results: DNA bands of the same size and position as the DNA ladder/ marker DNA
END-POINT ANALYSIS OF PCR
a laboratory technique that detects RNA in a biological sample, such as blood or tissue, to study gene expression
purified RNA fragments from a biological sample (such as blood or tissue) are separated by using an electric current to move them through a sieve-like gel or matrix, which allows smaller fragments to move faster than larger fragments.
Northern Blot (RNA)
Northern Blot (RNA) process
RNA extraction
electrophoresis
RNA separated by size
Northern blotting
labeled probes (binds gene of interest, complement)
Visualization of labeled RNA on X-ray film or fluroscent labeled
END-POINT ANALYSIS OF PCR
a method for hybridizing one or more labeled DNA probes to a large number of DNA fragments and discriminating among them. The procedure depends on the ability of denatured DNA single strands to bind tightly to nitrocellulose under certain conditions.
Southern Blot (DNA)
Southern Blot (DNA) process
Nitrocellulose filter on PCR samples
Blotting paper to dry
the DNA transferred to filter
then Hybridize with unique nucleic acid prob to be a complementary DNA sequence
Expose XRAY to filter
autoradiogram
END-POINT ANALYSIS OF PCR
is a method for determining the sequence of DNA nucleotides in a specific region of the genome. It’s also known as the “chain-termination method”.
Sanger Sequencing
Sanger Sequencing process
denaturing dsDANA with heat
making multiple copies of a segment
add primers
add to 4 polymerase solutions
add complimentary chain on segments until termination
denature the grown chains and eletrophorese
Sanger Sequencing 4 polymerase solutions
- ddATPS, dATPs, dCTPs, dGTPs, dTTPs
- ddTTPs, dATPs, dCTPs, dGTPs, dTTPs
- ddCTPs, dATPs, dCTPs, dGTPs, dTTPs
- ddGTPs, dATPs, dCTPs, dGTPs, dTTPs
