PCR and DNA Fingerprinting

What does a Basic Toolkit to mainpulate DNA require?

• Visualisation​

• Sequencing​

• Copying and modifying​

What does Gel Electrophoresis do?

Separates a mixture of molecules according to size

How does DNA’s charge affect Gel Electrophoresis?

DNA has -ve charge, attracted by +ve pole in electric field

Charge is proportional to length/mass of DNA, so acceleration is the same. How is DNA still able to move in Gel Electrophoresis?

Moves through gel so smaller molecules move faster

What is the ‘EB’ DNA Binding Fluorescent Dye?

Ethidium Bromide, intercalating agent

What is the ‘SG’ DNA Binding Fluorescent Dye?

SYBR Green, minor groove binding, only interacts w/ dsDNA

How does Ethidium Bromide work?

Excited w/ blue light, emits orange light

What are the steps of Energy Excitation?

• Excitation

• e(-) promoted to higher state

• e(-) returns to lower state

• Fluorescence emitted

What are used as Size Standards/Molecular Weight Markers in Gel Electrophoresis?

Bands of known size

What is the relationship of light and mass in Gel Electrophoresis?

Brightness correlates w/ mass of dsDNA proportional to amount of fluorophore intercalated trail

What is the importance of the vertical position in Gel Electrophoresis?

Gives relative size

What does PCR do?

Produces copies of a specific target segment of DNA in vitro

How does DNA Polymerase make PCR possible?

Several DNA Polymerases isolated

How can DNA Polymerases be an issue for PCR?

Synthetic DNA Polymerase needed

How can Primers be an issue for PCR?

Primers needed, need to be synthesised to bind where you want

How can information be an issue for PCR?

Need info about sequence you want to amplify

What are the 3 steps of the PCR Cycle?

Denaturation, Annealing, Extension

What are the products after 1 PCR cycle?

2 copies of each strand, grows exponentially 2^x

How many Primers are needed each PCR Cycle?

1 Primer

Describe the Sensitivity of PCR?

Sensitive, amplification means very little initial sample needed

Describe the Specificity of PCR?

Specific to an amplicon because of primers

What is an Amplicon?

Target segment

Describe the speed of PCR

Fast, each cycles takes around 2 mins

What is the relationship between the Amplicon and Speed in PCR?

Duration depends on amplicon length

What is the Knowledge Limitation of PCR?

Knowledge limited to known sequences so primers can be designed

Why are DNA-dependent Polymerases a limitation of PCR?

Require primers

How is Heat a limitation of PCR?

Denaturing step means thermoresistant polymerases needed

How is Length a limitation of PCR?

Longest amplicon depends on processivity of polymerase

How is PCR’s precision a Limitation?

Imprecise, common PCR polymerases have an error rate of 1/1000nt

How is using isolated DNA Polymerases a limitation of PCR?

Not entire replication machinery

What did Baltimore and Temin try to do?

Understand replication of tumour inducing viruses w/ RNA genomes

What was the product of Baltimore and Temin’s work?

Purified viral RNA-dependent DNA polymerase, Reverse Transcriptase

How is Complementary DNA made in Reverse Transcription PCR?

Reverse transcriptase added to mRNA to make cDNA

What is the purpose of cDNA in Reverse Transcription PCR?

Template for PCR amplification of gene of interest

What does Reverse Transcription PCR allow?

Testing for gene expression, finding alternative splicing isoforms

What does Quantitative Real-Time PCR allow?

To establish quantitative differences btwn samples

Non-coding sequences in Eukaryotes

Eukaryotic DNA contains huge amount of non-coding sequences

What are Short Tandem Repeats?

Simple sequence repeats

Tandem Repeats are Polymorphic, what does this mean?

Different across individuals

What is Polymorphism originated by?

Strand slippage during DNA Replication

What do Microsatellites’ high polymorphism allow?

Usage as genetic markers

What is the Genetic Profile?

Individual’s unique set of genetic markers

How can the Genetic Profile be obtained?

Analysis of tissue/body fluids, constituting DNA fingerprint

What is the High Certainty of Identification?

Probability of 2 people having same STR profile

What does STR stand for?

Short Tandem Repeats

How are PCR and STRs useful during mass casualties?

Help identify species

Role of PCR/SR in pregnancy?

Can be used for paternity tests

How do Ancestry Companies work?

Offer genetic testing/data analysis to build genealogies often w/ non-STR loci

What is the easiest type of old DNA for PCR to work on?

Mitochondrial, many copies per cell

What is the challenge using PCR w/ old DNA?

Contamination w/ modern DNA