Lecture 17 - 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

Transcription

Synthesis of RNA from a DNA template

Gene Expression

Process by which genetic information is used to produce proteins

RNA Polymerase

Enzyme that synthesises RNA from DNA

RNA Polymerase Function

Unwinds DNA, reads template, synthesises RNA 5′→3′

Template Strand

DNA strand used to synthesise RNA

Coding Strand

DNA strand with same sequence as RNA (T→U)

Uracil (U)

Base used in RNA instead of thymine

No Primer Required

RNA polymerase can initiate synthesis without a primer

Promoter

DNA sequence where RNA polymerase binds to initiate transcription

Transcription Unit

DNA sequence that is transcribed into RNA

Terminator

DNA sequence that signals end of transcription

Initiation (Transcription)

RNA polymerase binds promoter and unwinds DNA

Elongation (Transcription)

RNA polymerase synthesises RNA strand

Termination (Transcription)

RNA polymerase detaches at terminator

Transcription Rate

~60 nucleotides per second in E. coli

Multiple Transcription

Several RNA polymerases can transcribe a gene simultaneously

Operon

Cluster of genes regulated together under one promoter

Operator

DNA sequence acting as on/off switch for operon

Lac Operon

Regulatory system controlling lactose metabolism genes

lacZ

Gene encoding β-galactosidase

lacY

Gene encoding permease

lacA

Gene encoding transacetylase

LacI Protein

Repressor protein that inhibits lac operon

Repressor

Binds operator to block transcription

Inducer

Molecule that inactivates repressor (e.g. allolactose)

Lac Operon OFF

Repressor bound, transcription blocked

Lac Operon ON

Inducer binds repressor, transcription occurs

Allolactose

Molecule that inactivates lac repressor

Positive Regulation

Enhancement of transcription (e.g. cAMP-CRP)

cAMP

Cyclic AMP increases when glucose is low

CRP Protein

Binds cAMP and enhances RNA polymerase binding

Glucose Effect

High glucose inhibits lac operon activation

Combinatorial Regulation

Gene expression controlled by multiple signals

PaJaMo Experiment

Showed lacI encodes a repressor, not inducer

Constitutive Expression

Gene expressed continuously due to lack of repressor

Prokaryotic Transcription

Occurs in cytoplasm and can be simultaneous with translation

Eukaryotic Transcription

Occurs in nucleus, separate from translation

Pre-mRNA

Primary RNA transcript in eukaryotes

5' Cap

Protects mRNA and helps ribosome binding

Poly-A Tail

Adds stability and aids export of mRNA

Splicing

Removal of introns from pre-mRNA

Introns

Non-coding regions removed during splicing

Exons

Coding regions retained in mature mRNA

Spliceosome

Complex that removes introns

Alternative Splicing

Produces different proteins from same gene

TATA Box

Key promoter sequence in eukaryotes

Transcription Factors

Proteins regulating transcription initiation

Mediator Proteins

Assist binding of transcription machinery

Gene Regulation

Control of when and how much genes are expressed

Multicellularity Requires Regulation

Different cells express different genes