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Western University
Biochemistry 2280A
Eric Ball

Brandl – Lecture 8 Notes DNA Fingerprinting:  Method for identifying individuals and is done through comparison of multiple hypervariable regions in DNA  There are dinucleotide repeats within our genome of (CA) where n = 4  40 o Note that n varies in different individuals  Such repeats occur (with different flanking sequences) about 100,000 times in the human genome - are called VNTRs (variable number of tandem repeats)  To “fingerprint” an individual, you use primer pairs surrounding a number of these repeats VNTRs (or STRs):  In many regions of our genome, there are repetitive sequences (CACACACA…)  The repeat varies from ~4-40 times depending on the allele  VNTR – the repeat sequences are called VNTRs for Variable Number of Tandem Repeats (or STRs for short tandem repeats)  Each VNTR is amplified by PCR and analyzed by agrose gel electrophoresis  PCR products with differing numbers of repeats will run at different positions on the gel  The profile for all the repeats analyzed represents the fingerprint for that individual  DNA fingerprinting by PCR is such a powerful tool for forensics because of its sensitivity - extreme care must be taken not to contaminate samples  It is based on probability when looking at the same or multiple loci – two individuals could have the same pattern by chance (chance is not totally random though – some people in the populations may have a certain bias)  The amount of DNA required for fingerprinting can be as little as that found in a single hair follicle Our Genome is Diploid:  Will see two bands  Genome is diploid so that in general, the PCR from one locus will give two bands from each gene – one maternal copy and one paternal copy Different Sequences, representing the sequence on either side of the VNTR Note…  Two individuals might have the same 2 bands simply by chance  For this reason multiple VNTR loci are analyzed in a DNA fingerprint  Each VNTR locus requires a distinct pair of primers CSI London: 1. You are a member of a CSI unit investigating a robbery 2. From the scene you carefully remove a hair that may be the culprit’s 3. The crook was probably wearing a mark but witness noticed the get away card 4. You track down 3 possible suspects and obtain biological sample from each A, B, C 5. In the lab you isolate DNA from the forensics and suspect samples 6. You do PCR with each of the DNA that is isolated from the suspects with the forensic DNA sample 7. You analyze the PCRT products by agrose gel electrophoresis Genomic and cDNA Libraries:  Until the advent of PCR, isolation of genes requires their selection from pools or “libraries” that represented the full collection of genes for an organism  Each fragment, often of approx 10,000bp is cloned individually (without knowing what it contains) into a plasmid vector  The usual approach for cloning the genomic DNA randomly is to digest genomic DNA to less than completion (partial digest) with a restriction enzyme  The plasmid vector can then be cut with the same enzyme to allow the ligation of the genomic fragments How many independent clones of ~10, 000bp would be necessary to cover the full human genome?  If you desire the whole gene (introns/exons) or if you want promoter sequences, a genomic library is required  Other applications required only the coding regions – for example, if you want to express the protein in bacteria – use a cDNA library which represents all of the mRNAs found in one cell type (c = complementary, i.e. complementary to the mRNA) What is cDNA?  The cDNA is a replicate DNA copy of mRNA (c = complementary)  The cDNA can be cloned (or can be used in PCR)  The cDNA libraries from different cell types will differ dramatically as the cells express different mRNAs  There are no introns in cDNA – cell has spliced out the introns to make the mRNA o If use genes in E.coli, the E.coli couldn’t handle the introns so we isolate RNA from the cells after splicing, and then make cDNA from that  The process of cDNA library construction takes advantage of the enzyme reverse transcriptase, which synthesizes single stranded DNA from a RNA template Making cDNA:  Isolate RNA from a tissue  The mRNA has the polyA tail  We want to make a complementary strand to that message – need the presence of dNTPs  Need a primer which will anneal to the polyA tail very conveniently  Introduce an enzyme called reverse transcriptase which will transcribe the RNA into DNA  DNA copy of an RNA template  Naturally found in certain viruses which have RNA genomes  Making double stranded o Have to degrade the RNA from rRNase to make it double stranded o Make second strand by adding dNTPs using DNA polymerase, and get a second copy of the DNA and now you have duplex DNA o Some types of RNA fragments acts as primers o Double stranded cDNA copy of original mRNA Expression of Cloned Genes:  In many instances the end product in which we are interested in is not the gene
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