Gene & Genomes - Lec 2 - How to sequence genome Flashcards
Learn how a genome is sequences
what is the Human Genome Project Goal
a) read the entire script of our sequence in the corresponding order
b) Identifying all human genes
C) lay out a foundation for sequencing of other species
what are the three major problems for genome sequencing
1) Genomes are really big
2)They have lots of repeated bits
3) Need a lot of DNA to get sequence
What are the solutions to problems for genomes sequencing
1) cut the DNA into smaller pieces that can be put back together
2) take lots of copies of each bit
What does PCR enable us to do?
Large amount of DNA to be produce from small/complex samples
What does PCR use to sequence DNA
- Two primers & DNA polymerase
- Bases
-Buffer - Thermal cycler
What temperature is the thermal cycler on?
95 degrees - break down DNa
55 degrees - allow primers to anneal to DNA
72 degrees - Optimum temperature for DNA polymerase
What does gel electrophoresis enable as to do with PCR results?
Separates charges DNA molecules, to be seen when stained with fluorescent dye under UV light.
What was done to reduce the complexity of PCR?
1) Standard limit of around 3000Bp
2) Design primers
What is Sanger sequencing?
Uses one single primer and polymerase to make a new single strand of DNA but terminates at bases.
What is done in Sanger sequencing to enable an electrophoresis machine to chart out the DNA sequence by laser?
The DNA bases are labelled with different coloured tags, separated by size/colour.
How do you reduce the complexity of a genome to sequence 600-1000Bp?
By reducing the complexity of a genome using restriction enzymes.
What are restriction enzymes?
Enzymes that recognise and “cut” specific sequences and nowhere in an ordered maner.
How is fragmented restriction digested DNA separated?
Bacteria/cloning is used, in other words vectors
What 2 purposes do vectors serve?
1) Separate fragments so they can be sequences individually
2) Allow production of lots of DNA by growing up lots of bacteria containing BACs/Plasmids
Explain the use of Bacterial artificial chromosomes in 5 steps.
The primary method used for BAC-based sequencing.
1) Chromosomes are fragmented into 150,000-200,000 Bp.
2) Each BAC is mapped to determine where in the DNA the BAC clone comes from in the genome
3) Each back clone is cut into smaller 2000bp fragments
4) Each fragment sequences around 10 times
5) Computer analysis assembles the sequences into a continuous stretches of sequence representing the BAC clone
How is the physical location of BACs mapped using in situ hybridisation?
Hybridising them to chromosome spreads
What is shotgun sequencing?
Shatters DNA sequence from individual BACs into smaller fragments. assemble DNA from smaller matching sequences using bioinformatics
What is a contig & Scaffold in shotgun sequencing?
Contig: Continuous length of DNA covered by overlapping reads
Scaffold: a genome assembly composed of contigs and gaps
Explain shogun sequencing step by step.
Fragment BACs/chromosomes -> Sequence individually
-> Find overlaps using computing -> Find longer sequence using fragments
what is a structural annotation?
- Identifying the genes structure, intron & exon.
- Location of regulatory motifs
What is Functional annotation?
attaching biological information to genomic elements: Biochemical function, biological function, regulation, interaction, expression
How do we identify genes in a genome?
1) homology
2) Mapping of the transcriptome
3) Automated annotation
What is Homology
Use of knowen gene sequence from another (closely) related species to identify similar sequences in species of intrest
What are auto annotations?
Gene start with ATG and end with TAA, TAG or TGA
