DNA Sequence Organisation of the Human Genome Flashcards
Explain how DNA was shown to be the genetic material Describe some DNA analysis techniques Recognise the key DNA sequence features in the human genome including their arrangement and significance
How did Fred Griffiths discover that DNA is the genetic material?
2 different strains of bacteria. Strain S was pathogenic and smooth because of mucus coating. Strain R rough and non pathogenic. Heat killed S cells were non pathogenic, but a mixture of these and R cells were pathogenic, which means that the R strain acquired S type characteristics, via genetic material - later found to be DNA.
How did later experiments confirm that DNA was the transforming principle?
They separated heat killed S cells into DNA proteins lipids and carbs, and only R cells mixed with DNA became pathogenic, which means they took up the DNA and acquired S cell characteristics. Showed that we could use DNA to permanently and precisely modify the genetics of an organism.
Explain Sanger Sequencing and how it can be used to analyse DNA.
Requires target DNA for sequencing, oligonucleotide primers (20nt) complimentary to a prt of the target, DNA Polymerase for extending the primer, and a mixture of dNTPs and ddNTPs. ddNTPs lack a 3’-OH so they cannot be extended by DNA Polymerase, which leads to a chain termination. As the process goes on, a ddNTP is incorporated into every possible location in the chain. The fragments are separated by electrophoresis. ddNTPs can be different colours which allow a direct read of sequences up to 1k nt.
Explain what restriction enzymes are and how they can be used to clone DNA.
They are endonucleases which cut dsDNA at specific sequences, leaving 3’-OH and 5’-P groups. Endonucleases cut internal bonds and circular DNA. They require 4-8nt restriction sites. They originally come from bacteria and target viral DNA. If the restriction site is palindromic, the cut will leave sticky ends, if not it will leave blunt ends. For DNA cloning, both the target gene and the plasmid are cut by the same restriction enzyme so that the sticky ends are complimentary. Target ligated into plasmid, plasmid goes into bacteria and then bacterial replication leads to cloning of the gene.
What is Southern Blotting?
A way to visualise DNA on a membrane. A mixture of DNA fragments is ran through an agarose gel via electrophoresis and then transferred onto a nylon membrane. Radioactive/fluorescent DNA probes hybridise to the complimentary fragment and allow visualisation. Northern Blotting is similar but for RNA.
Explain the process of PCR.
Allows the generation of ug of DNA fragment (up to 10kB) from only ng. A heating step separates the DNA into two strands, oligonucleotide primers hybridise next to the target sequence in the cooling step, and then are extended by Taq Polymerase (heat stable) which adds dNTPs to 3’OH of each primer. The target sequence is duplicated in each cycle which means there are 2^n fragments after n cycles.
Why is PCR useful?
Allows for quicker detection of target sequences than cloning using restriction enzymes or detection by blotting. PCR can also determine the presence of a target sequence in a mixed sample. The products of PCR can be purified and used for downstream experiments are qPCR allows for determination of the amount of target before amplification.
Describe how NGS works
A DNA sample is fragmented and each fragment attached to adaptor sequences. The library is attached to a flowcell via oligonucleotides and bridge amplification takes place which builds up a cluster of DNA fragments all originating from one fragment. Nucleotides are added which are reversibly tagged and blocked, and after incorporation the fluorescence is laser detected, and the tags and blocks cleaved off and the process carries on. The sequence is compared to a reference genome.
What are the applications of NGS?
Can be used to sequence a whole genome - detecting variation between individuals. Targeted sequencing - exome sequencing reveals protein coding variations. Transcriptome sequencing (RNAseq) - DNA reverse transcribed from RNA. The amount of reads is indicative of the level of expression.
Describe the structure of protein coding DNA and how it compares to other organisms
1200Mb of DNA is genes and gene related, only 48Mb exons. Most of the gene is non-coding due to 5’/3’ UTR, enhancer/promoter regions and long introns. Plants have many more protein coding genes, and nematode worms have the same. However, the phenotypic complexity of humans comes from alternative splicing - one gene resulting in multiple mRNA isoforms and hence proktein isoforms. The average human gene is 27Kb with 10.4 exons of size 178bp. Mean intron length is 5.4Kb. 25% of genome consists of gene deserts - over 1Mb no genes.
What is non-coding RNA and what are the different types?
75% of the genome is transcribed to RNS, and about 1/3 is non-coding RNA, which means it's not translated into protein. Some of it is transcriptional noise but some of it has known function. Ribosomal RNA genes Transfer RNA genes Small nuclear & nucleolar RNA genes MicroRNA genes lncRNA
Explain the structure and function of Ribosomal RNA genes
It’s essential for translation and consists of 4 types, 18S, 5.8S, 28S & 5S. The genes exist in multiple copies to ensure that there are enough. All apart from 5S are derived from a 41S precursor which is transcribed by RNA P1 from about 300 copies of a gene which is tandemly repeated in 5 clusters on different chromosomes. Similar for 5S rRNA but located elsewhere in genome and transcribed by RNA P3.
Explain the structure and function of tRNA genes
Deliver amino acids to the ribosome. 76-90 nt with a folded structure. Genes for the 49 different tRNAs exist as multiple copies at various chromosome sites are transcribed by RNA P3.
Explain small nuclear and small nucleolar RNA genes.
Dispersed throughout the genome and transcribed by RNA P2/3. Each gene makes a distinct RNA with a distinct processing function for mRNA, tRNA, or rRNA into their mature forms. Many are essential components of the spliceosome.
Explain the structure and function of microRNA genes
About 22nt RNAs which modulate gene expression by RNA interference (physcial blocking of the mRNA for ribosomal access). Different miRNAs for different mRNAs are transcribed from different genes by RNA P2/3. Piwi interacting RNA (piRNA) are similar but work in germ cells by silencing transposons.
