THEME 4 MOD 3 Flashcards
Kary mullis, 1985
developed PCR technique: – polymerase chain reaction
- amplify dna in a tube up to millions of copies
How has PCR been used in research
- diagnoses of genetic defects
- detection of viral dna
- amplifying trace dna in fossils
- forensic investigations : link individuals to samples
describe PCR
- dna in a buffered solution in a tube with dNTPs (free deoxyribonucleotides), ions, salts, pair of short primers (15-30 nucleotides long) which anneal to specific parts of dna and allow for replication from that point
- thermocycling process breaks hydrogen bonds between complementary dna strands, PCR only needs enzyme for synthesis of daughter strands
- taq polymerase is added to the tube (taken from thermus aquaticus bacteria, is heat tolerant up to 95 degrees) and can catalyze synthesis of daughter strands
3 stages of PCR reaction
denaturation, annealing, extension
PCR
chain reaction that brings about a exponentially growing population of a target sequence of identical dna molecules
Describe the steps of PCR
- researcher designs a primer that will anneal to will anneal to a complimentary sequence near their gene of interest.
- Denaturation: DNA double helix unwound by thermocycler heating the solution to break hydrogen bonds between base pairs of the complementary strands
- thermocycler cools the solution so the rna primers can anneal to the complementary sequence on the two dna strands
- Extension: taq polymerase extends and polymerizes the daughter strands from the primers in 5’ to 3’ direction
- Now have two double stranded helices of dna containing desired target sequence portion of template dna
how many copies of dna per number (n) of pcr cycles
n^2 dna copies of the target sequence for each round of PCR
after many rounds the dna will be exponentially amplified millions of times to allow for applications like genetic testing or forensics
How can dna target sequences amplified by pcr be visualized
gel electrophoresis:
seperate dna or other molecules based on their rate of movement through agarose gel in an electric field
describe gel electrophoresis
dna is loaded into wells of a pouros gel in an electric field with a positive and negative side. Because dna is negative due to the sugar phosphate back bone, dna will move through the gel towards the positive end. DNA segments will move at different rates depending on their size, smaller segments will move faster through the gel than large molecules.
gel electrophoresis visualizing dna of different sizes
- gel electrophoresis can separateWh dna segments from 100s to 10,000 nucleotides in length
- a standardized ladder of dna of standardized lengths is also loaded into the gel
- dna can be stained so it fluoresces under UV
- after the electrophoresis process, DNA bands of different sizes can be seen and identified under UV
Who developed DNA sequencing and why is it important?
Frederick sanger, 1975
can determine the sequence of a dna molecule to understand and identify coding and noncoding regions
could only determine the sequence of small segments
What is shotgun sequencing
- created by gene myers and jim webber
- break genome into different sized pieces
- Phase 1: random sequencing of dna of each fragment
-Phase 2: identify overlap between generated fragments and assemble a long continuous strand of nucleotides in DNA that make up each chromosome.
-Phase 3: annotate the sequences of dna for to identify regulatory regions, genes, and non coding regions
What was the timeline of whole genome sequences done
needed computational software capable of facilitating assembly of fragments to be able to sequence the whole genome of an organism
1995: haemophilus influenza
1998: C. elegans, first multicellular genome
1999: Drosophila
2000: entire human genome
dideoxy chain termination method / dideoxy sequencing / sanger sequencing technique
used in earlier approaches to dna sequencing
- dna to be sequenced serves as template strand
- need components of dna replication: short single stranded dna primers, sufficient free deoxyribonucleotides, and dna polymerase
-also need smaller amount of modified deoxyribonucleotides (ddNTPs) without the OH on 3’ end which would interupt daughter strands at varying lenths
- theorized that with enough ddNTPs there would be daughter strands terminated at every nucleotide in the sequence
visualizing dna after dna sequencing (dideoxy sequencing)
- each ddNTP fleourescently labelled a different colour to visulaize all the different terminators in a sequence of dna
- gel electrophoresis preformed on the samples, and continues until a laser excites the fluorescent dye in every dna band
- strands differing as little as one nucleotide in length can then be detected by fluorescence detector which reads the fluorescence level, records it, and matches it to one of four ddNTPs
- spectrogram trace then maps fluorescent levels, with each peak corresponding to the nucleotides complementary to the template strand
- the order of fluorescently tagged nucleotides can be read as the complementary dna sequence from left to right
why is shotgun sequencing advantageous over dideoxy chain termnination?
- dideoxy sequencing can only determine short dna sequencse up to a few hundred nucleotides in length
- shotgun sequencing allows for multiple dna sequences to be examined for areas of overlap (or contigs) and organized into a long continuous sequences, sometimes making up a large part of a chromosome, being millions of nucleotides in length
- algorithms assemble the continuous sequence through automated computer programs
- sequences of genomes can be determined through areas of overlap of sequenced dna segments
annotation of dna sequences
- identify specific regions of interest and examine series of nucleotids to better understand how they code for our genome
- need to getermine the correct reading frame: there are 6 possible reading frames (3 per dna strand)
- computer software scans sequence to determine reading frame that works for both strands, generally a long sequence without stop codons indicates a good reading frame (likely a coding region)
in examining beta globin protein sequence, only one frame will give us the fully functional protein sequence - computer software looks for gene-sized stretches of nucleotides with no internal stop condons and flanked by a start and stop codon of either end, will also scan for promoters and regulatory regions
How can intron and exon regions be identified in annotation of an eukaryotic genome
- identify patterns (aka sequence motifs), like open reading frames (stretch with no stop codon), triplets of nucleotides that code for the specific amino acids of a protein, transcription factor binding sites within, upstream or downstream of intron
- can identify some sequnce motifs by the hypothetical rna, as a sequence with nearby complimentary regions to each other could be identified as a trna, as these molecules form base pairs with themselves and fold upon themselves into hairpin loops
what is the purpose of repeat sequences that do not code for anything
- approx. 50% of eukaryotic genome consists of repeat non coding sequences
- encode noncoding functional and noncoding rna sequences
- non coding sequences vary from individual to indivdual, and between species, contributing to genetic diversity and diversity across organisms
