Human Genome Project History

Human Genome Project & PCR History

• Started in 1990, involving around 30 different institutions
• Goal: fully sequence the human genome
  • Sequence the chemical bases making up human DNA
  • Identify all human genes
  • Develop tools for genetic information analysis
• Importance:
  • Helped scientists understand, prevent, and treat diseases
  • Led to development of new technologies and analytical tools
  • Established an open data-sharing approach
  • Advanced policies and support for scientific data sharing
  • Raised awareness on ethical considerations
• Impact on medicine:
  • Established somatic cell genetic disease as a category
  • Helped define mutation as the basis of cancer
  • Altered medical practice
• Impact on other areas:
  • Inspired other large-scale data acquisition like the International HapMap Project and The Cancer Genome Atlas
• Initial completion in April 2003
• Mission Accomplished in May 2021
• Total genome size: approximately 3.2 billion base pairs
• Challenge: numerous other sequences exist in the genome not of interest
  • Specificity is key as the genome is large
• DNA amount in the samples of interest is very small
  • Amplification is crucial as DNA samples are often limited
• Inefficiency in studying specific sequences due to specificity and amplification issues is resolved through polymerase chain reaction.
• Dr. Kary Mullis discovered that doubling the test target DNA yields 21 copies
  • Repeating N times results in 2N
  • Adding an oligonucleotide primer with the 4 dNTPs and DNA polymerase in a chain reaction allows millions of copies to be made (N = 30 or 40)
  • Kary B. Mullis who won the Nobel Prize in Chemistry in 1993 for the easy and cheap analysis of DNA
  • Led to advancements in diagnostics, molecular biology and forensics

Polymerase Chain Reaction (PCR) Steps

• PCR amplifies DNA

PCR Cycles

• First cycle:
  • Denaturation: double-stranded DNA is heated to separate into single strands
  • Annealing: primers bind to specific complementary sequences on single-stranded DNA
  • Extension: DNA polymerase synthesizes new DNA strands using primers as starting points
  • A total of two copies after first cycle
• The newly synthesized strands then also serve as templates for another round of PCR
• The process repeats leading to 4 copies of the target DNA sequence
• In the third cycle, the cycle repeats and produces 8 copies
• DNA strands accumulate exponentially
• Process continues for multiple cycles, around 30 in a standard PCR reaction
• By the 30th cycle, approximately copies of the target DNA sequence are generated, demonstrating the power of PCR in DNA amplification
• Exponential DNA replication is achieved through denaturation, annealing and extension

Denaturation & Annealing

• Denaturation unfolds or breaks up protein by modifying its standard three-dimensional structure Proteins can be denatured by chemical action, heat, or agitation, causing protein to unfold or it polypeptide chain
• Denaturation typically leaves molecules non-functional
• Double-stranded DNA separates into two single strands at 94 - 96°C in several seconds to minutes
  • Longer Template - longer time
• Annealing:
  • A most critical step for specificity
  • Primers hybridize to their complementary DNA sequences
  • Temperature ranges from 50-70°C
  • A starting point is determined using the melting temperature (Tm) of the primer sequences
• Melting Temperature (Tm): temperature at which half double-stranded DNA exists as single-stranded DNA
  • Affected by: reaction conditions, salt concentration, mismatches, template condition and secondary structure

Importance of Melting Temperature

• Annealing: reaction temperature needs to be low enough for primers to bind DNA templates, but high enough so undesired duplexes do not form
• Amplification: all primers should have similar Tm values for similar temperature to anneal and disassociate for amplification
• Sequence Verification: melting analysis after PCR can monitor duplex hybridization during temperature change

PCR Components

Primers

• Single-stranded DNA fragments that are 20-30 bp long
• Act like primers in vivo
• Determine the specificity of PCR
• Have sequences complementary to sites flanking the region of interest

Forward & Reverse Primers

• Forward primer:
  • 5’ to the sequences amplified
  • Hybridizes with the minus strand
• Reverse primer:
  • 3’ to the sequences amplified
  • Hybridizes with the plus strand
• Should be forward to create a copy that is the same as the plus-strand sequence
• Primer Sequence affect efficiency in binding to the target via %GC and length of strand
• Melting temp (Tm) of the forward and reverse primer must be similar

DNA Template

• DNA Template from nucleotide extraction
• Routine clinical analyses require 100 ng – 1 µg
• Best templates are in good condition, free of contaminants, and have no nicks or breaks

Deoxynucleotide Bases

• Building blocks of DNA are
  • dATP (adenine)
  • dTTP (thymine)
  • dGTP (guanine)
  • dCTP (cytosine)
• Usual requirements: 0.1-0.5 mM of each

DNA Polymerase

• First Polymerase from E. coli
  • Needed to be added after each round of denaturation
• Taq Polymerase
  • Thermus aquaticus bacteria
  • thermostable
  • good fidelity
  • accurate DNA copying ability
  • Thermus aquaticus species is a bacteria that can tolerate high temperatures
• Taq polymerase belongs to the enzyme that synthesizes new DNA strands, adding nucleotides to the template
• Phase occurs at higher temperatures where most other enzymes would denature, making its heat-resistant activity crucial for PCR amplification
• Heat-stable enzyme extracted from "Thermus aquaticus": Commonly used in the PCR procedure to amplify specific DNA, able to function at high temperatures with no denaturation
• *Tth polymerase (Thermus thermophilus)
  • also has reverse transcriptase activity
  • also used in RT-PCR (RNA template)
• Gram - bacteria: Used in numerous biotechnological applications like structural genomics, or model for genetic manipulation